SE640 User Manual – English

SE640 User Manual – English
user manual
Hoefer SE640
Wide-mini Dual Gel Electrophoresis Unit
um SE640-IM/Rev.B0/07-12
Contents
Important Information . .......................................... ii
Waste Electrical and
Electronic Equipment (WEEE)................................vii
el electrophoresis unit
G
function and description.........................................1
Specifications........................................................2
Unpacking and inventory.........................................4
Operating instructions.............................................7
Care and maintenance..........................................23
Customer service information.................................24
Troubleshooting....................................................25
Appendix A: Laemmli system gels..........................29
Solutions ............................................................31
Gel recipes .........................................................34
Appendix B: Bibliography......................................36
Ordering information.............................................38
• pi
Important Information – English
• If this equipment is used in a manner not specified by Hoefer, Inc. the protection provided by the
equipment may be impaired.
• This instrument is designed for indoor laboratory
use only.
• Only accessories and parts approved or supplied
by Hoefer, Inc. may be used for operating, maintaining, and servicing this product.
• Rozeslat pouze voda nebo 50/50 voda/ethylenglykolu prostřednictvím výměník tepla je li to vybavena. Nemají připojení výměník tepla s vodními
setřepná nebo jakékoli chladicí kapaliny zdroje, kde
tlak vody je neregulo.
• Nikdy zavést prostředek proti zamrznutí nebo
jakákoli organická rozpouštědla do jakékoli části z
tohoto nástroje. Rozpustidlům způsobí nenapravitelné poškození jednotka!
• Only use a power supply that is CE marked or
safety certified by a nationally recognized testing
laboratory.
• Nejsou provozována s pufru teplotách nad
maximální stanovenou technickými specifikacemi. Přehřátí způsobí nenapravitelné poškození
jednotka!
• The safety lid must be in place before connecting
the power supply leads to a power supply.
Vigtig Information – Danish
• Turn all power supply controls off and disconnect
the power leads before removing the safety lid.
• Circulate only water or 50/50 water/ethylene glycol
through the heat exchanger if so equipped. Do
not connect the heat exchanger to a water tap or
any coolant source where the water pressure is
unregulated.
• Never introduce antifreeze or any organic solvent
into any part of the instrument. Organic solvents
will cause irreparable damage to the unit!
• Do not operate with buffer temperatures above
the maximum specified technical specifications.
Overheating will cause irreparable damage to the
unit!
Duležité Informace – Czech
• Pokud by toto zařízení je použito způsobem, který
není podle Hoefer, Inc. ochrana poskytovaná na
základě zařízení může být narušena.
• Tento nástroj je určen pro vnitřní použití v
laboratoři pouze.
• Pouze příslušenství a části schválen, nebo poskytnutých Hoefer, Inc. mohou být použity pro provoz,
údržbu, a údržbě tohoto výrobku.
• zdroj napájení používají jen že je opatřen
označením CE osvědčena nebo bezpečnost
vnitrostátně uznanými zkušebními laboratoř.
• Bezpečnosti lid musí být zavedena před připojením
napájecí zdroj napájení vede k.
• Turn veškeré napájení kontroly vypnuto a odpojit
před odběrem energie vede bezpečnostní víko.
• pii
• Hvis dette udstyr bruges i en måde ikke specificeret ved Hoefer, Inc. den beskyttelse, som er blevet
forsynet af udstyret kan måske svækkes.
• Dette instrument er designet for indendørs laboratoriumbrug bare.
• Bare tilbehør og del godkendede eller forsynede
ved Hoefer, Inc. kan måske bruges for drive, funktionsfejl, og betjening dette produkt.
• bruger Bare en strømforsyning, der er CE
markerede eller sikkerhed, som er blevet attesteret
af en, som nationalt er blevet anerkendt prøve
laboratorium.
• Sikkerhedlåget må være på plads før forbinding
strømforsyningsblyet til en strømforsyning.
• Drejer alle strømforsyningskontroller af og afbryder
kraftblyet før fjerning sikkerhedlåget.
• Cirkulerer bare vand eller 50/50 vand/ethylene
glykol gennem varmeveksleren i så fald udrustet.
Forbind ikke varmeveksleren til en vandhane
eller nogen kølemiddelkilde hvor vandtrykket er
unregulated.
• Introducerer Aldrig antifreeze eller noget organisk
opløsningsmiddel ind i nogen del af instrumentet.
Organiske opløsningsmidler vil forårsage uboelig
skade til enheden!
• Driver ikke med stødpudetemperaturer over
maksimummet specificerede tekniske specifications. Overheding vil forårsage uboelig skade til
enheden!
Belangrijke Informatie – Dutch
• Indien deze uitrusting in een manier wordt
gebruikt die niet door Hoefer, Inc. is gespecificeerd
de bescherming die door de uitrusting is verzorgd
kan worden geschaad.
• Dit instrument is voor binnenlaboratoriumgebruik
enkel ontworpen.
• Enkel onderdelen en delen keurden goed of
leverden door Hoefer, Inc. kan voor het bedienen
worden gebruikt, handhavend en onderhouden
van dit product.
• gebruik Enkel een netvoeding die CE is markeerde
of veiligheid die door een is gecertificeerd die
nationaal is herkend testene laboratorium.
• Het veiligheidsdeksel moet in plaats voor het
verbinden van de netvoeding leidt tot een
netvoeding zijn.
• Doe alle netvoedingscontroles Uit en koppel los
de machtleiding voor het verwijderen van het
veiligheidsdeksel.
• Circuleer enkel water of 50/50 water/ethyleenglycol door de hitte exchanger zo ja uitrust.
Verbind de hitte exchanger naar een waterkraan
of koelmiddelbron niet waar de waterdruk niet
geregulariseerd is.
• Stel Nooit antivriesmiddel of organische oplosmiddelen in deel van het instrument voor. Organische
oplosmiddelen zullen onherstelbare schade aan de
eenheid veroorzaken!
• Bedien niet met buffertemperaturen boven het
maximum specificeerde technische specificaties.
Oververhittend zal onherstelbare schade aan de
eenheid veroorzaken!
on kansallisesti tunnustettnut testaaminen laboratoriota.
• Turvallisuuskansi täytyy olla paikallaan ennen
yhdistäminen käyttöjännitelyijyjä käyttöjännitteeseen.
• Kiertää kaikki käyttöjännitevalvonnat ja irrottaa
valtalyijyt ennen poistaminen turvallisuuskantta.
• Kiertää vain vesi tai 50/50 vesi/ethyleneä glycol
siinä tapauksessa varustetun lämmönvaihtimen
läpi. Älä yhdistä lämmönvaihdinta vesinapautukseen eikä jäähdytysnestelähteeseen, missä vesipaine on unregulated.
• Pakkasneste eikä orgaaninen liuotin välineen
osassa ei esitele Koskaan. Orgaaniset liuottimet
aiheuttavat korvaamattoman vahingon yksikköön!
• Ei käytä puskuria yllä olevia lämpötiloja enintään
määritetyillä teknisillä täsmennyksillä. Ylikuumeneminen aiheuttaa korvaamattoman vahingon
yksikköön!
Information Importante – French
• Si cet équipement est utilisé dans une manière pas
spécifié par Hoefer, Inc. la protection fourni par
l’équipement pourrait être diminuée.
• Cet instrument est conçu pour l’usage de laboratoire intérieur seulement.
• Seulement les accessoires et les parties ont
approuvé ou ont fourni par Hoefer, Inc. pourrait
être utilisé pour fonctionner, maintenir, et entretenir ce produit.
• utilise Seulement une alimentation qui est CET a
marqué ou la sécurité certifié par un nationalement reconnu essayant le laboratoire.
Tärkeää Tietoa – Finnish
• Le couvercle de sécurité doit être à sa place avant
connecter l’alimentation mene à une alimentation.
• Jos tätä varusteita käytetään tavassa ei määritetty
Hoefer, Inc. suojelu ehkäisty varusteille saattaa olla
avuton.
• Tourner tous contrôles d’alimentation de et
débrancher les avances de pouvoir avant enlever le
couvercle de sécurité.
• Tämä väline suunnitellaan sisälaboratoriokäytölle
vain.
• Circuler seulement de l’eau ou 50/50 glycol d’eau/
éthylène par l’exchanger de chaleur si si équipé. Ne
pas connecter l’exchanger de chaleur à un robinet
d’eau ou à la source d’agent de refroidissement où
la pression d’eau est non régulée.
• Vain lisävarusteet ja osat hyväksyivät tai toimitti
Hoefer, Inc. oheen ää voi käyttää käyttämiselle,
valvoalle, ja servicing tämä tuote.
• Vain käyttää käyttöjännitettä joka on CE merkitsi
tai turvallisuus joka on todistanut aidoksi ohi joka
• Ne Jamais introduire d’antigel ou du dissolvant
organique dans n’importe quelle partie de
l’instrument. Les dissolvants organiques causeront
• piii
des dommages irréparables à l’unité!
• Ne pas fonctionner avec les températures de
tampon au-dessus du maximum a spécifié des
spécifications techniques. La surchauffe causera
des dommages irréparables à l’unité !
Wichtige Informationen – German
• Wenn diese Ausrüstung gewissermaßen nicht
angegeben durch Hoefer, Inc. verwendet wird,
kann der durch die Ausrüstung zur Verfügung
gestellte Schutz verschlechtert werden.
• Dieses Instrument wird für den Innenlaborgebrauch nur dafür entworfen.
• Nur Zusätze und Teile genehmigten oder lieferten
durch Hoefer, Inc. kann für das Funktionieren, das
Aufrechterhalten, und die Wartung dieses Produktes verwendet werden.
• Verwenden Sie nur eine Energieversorgung,
die CE gekennzeichnet oder durch ein national
anerkanntes Probelaboratorium bescheinigte
Sicherheit ist.
• Der Sicherheitsdeckel muss im Platz vor dem
Anschließen der Energieversorgung sein führt zu
einer Energieversorgung.
• Alle Energieversorgungssteuerungen abdrehen
und die Macht trennen führt vor dem Entfernen
des Sicherheitsdeckels.
• Nur Wasser oder 50/50 Glykol des Wassers/
Äthylens durch den Wärmeaustauscher, wenn so
ausgestattet, in Umlauf setzen. Verbinden Sie den
Wärmeaustauscher mit einem Wasserklaps oder
jeder Kühlmittel-Quelle nicht, wo der Wasserdruck
ungeregelt wird.
• Führen Sie nie Frostschutzmittel oder jedes
organische Lösungsmittel in jeden Teil des Instrumentes ein. Organische Lösungsmittel werden
nicht wiedergutzumachenden Schaden der Einheit
verursachen!
• Mit Puffertemperaturen über angegebenen
technischen Spezifizierungen des Maximums
nicht funktionieren. Die Überhitzung wird nicht
wiedergutzumachenden Schaden der Einheit
verursachen!
Informazioni Importanti – Italian
• Se quest’apparecchiatura è usata in un modo
specificato da Hoefer, Inc. la protezione fornito
dall’apparecchiatura potrebbe essere indebolita.
• Questo strumento è disegnato per l’uso di laboratorio interno solo.
• Solo gli accessori e le parti hanno approvato o
hanno fornito da Hoefer, Inc. potrebbe essere
usato per operare, per mantenere, e per revisionare
questo prodotto.
• usa Solo un alimentatore che è CE ha marcato o la
sicurezza certificato da un nazionalmente riconosciuto testando il laboratorio.
• Il coperchio di sicurezza deve essere nel luogo
prima di collegare i piombi di alimentatore a un
alimentatore.
• Spegne tutto i controlli di alimentatore e disinserisce i piombi di potere prima di togliere il coperchio di sicurezza.
• Circola solo l’acqua o 50/50 glicole di acqua/etilene
attraverso lo scambiatore di calore se così equipaggiato. Non collegare lo scambiatore di calore a un
rubinetto di acqua o qualunque fonte di refrigerante dove la pressione di acqua è sregolata.
• Non introduce mai l’antigelo o qualunque solvente
organico in qualunque parte dello strumento. I
solventi organici causeranno il danno irreparabile
all’unità!
• Non opera con le temperature di tampone al di
sopra del massimo ha specificato le descrizioni
tecniche. Il surriscaldamento causerà il danno
irreparabile all’unità!
Viktig Informasjon – Norwegian
• Hvis dette utstyret blir brukt i en måte ikke spesifisert ved Hoefer, Inc. beskyttelsen som ha blitt git
av utstyret kan bli svekket.
• Dette instrumentet er utformet for innendørs laboratoriumbruk bare.
• Bare tilbehør og deler godkjente eller forsynte ved
Hoefer, Inc. kan bli brukt for drive, vedlikeholde, og
betjene dette produktet.
• bruker Bare en kraftforsyning som er CE merket
eller sikkerhet som ha blitt sertifisert av et som
nasjonalt ha blitt anerkjent prøver laboratorium.
• piv
• Sikkerheten lokket må være på plass før forbinding
kraftforsyningene blyene til en kraftforsyning.
• Vender all kraftforsyningsstyring av og frakopler
kreftene blyene før fjerning sikkerheten lokket.
• Sirkulerer bare vann eller 50/50 vann/ethylene
glykol gjennom oppvarmingen veksleren i så fall
utstyrer. Ikke forbind oppvarmingen veksleren
til en vanntapp eller noe kjølemiddelkilde hvor
vannet trykket er unregulated.
• Introduserer Aldri antifreeze eller noe organisk
løsemiddel inn i noe del av instrumentet. Organiske løsemiddler vil forårsake irreparabel skade på
enheten !
• Driver med buffertemperaturer over maksimum
ikke spesifiserte teknisk spesifikasjoner. Å overoppheting vil forårsake irreparabel skade på enheten !
Wazne Informacje – Polish
• Jeżeli ten sprzęt jest wykorzystywany w sposób nie
określone przez Hoefer, Inc. do ochrony przewidzianej przez urządzenie może zostać obniżony.
• Instrument ten jest przeznaczony do użytku w
laboratoriach kryty tylko.
• Tylko akcesoriów i części zatwierdzone lub dostarczone przez Hoefer, Inc. mogą być wykorzystane do
eksploatacji, utrzymania i obsługi tego produktu.
• korzystać jedynie zasilacza że jest noszące oznakowanie CE lub bezpieczeństwa uwierzytelnione
przez uznane na poziomie krajowym laboratorium
badawcze.
• Bezpieczeństwo lid musi być w miejsce przed
podłączeniem zasilania prowadzi do zasilania.
• Zaś wszystkie źródła zasilania urządzenia sterujące
off i odłączyć moc prowadzi przed odbiorem
bezpieczeństwa lid.
• Krążą tylko wody lub wody 50/50/ethylene glycol
wymiennik ciepła poprzez jeśli tak wyposażone.
Nie należy połączyć wymiennik ciepła woda z
kranu lub jakimkolwiek chłodziwo źródła, jeżeli
ciśnienie wody jest nieuregulowanych.
• Nigdy nie wprowadzać rozpuszczalnika organicznego przeciw zamarzaniu lub jakichkolwiek
na dowolną część dokumentu. Rozpuszczalniki
organiczne spowoduje nieodwracalne szkody dla
jednostki!
• Nie działają w buforze temperatury powyżej
maksymalnego określone specyfikacje techniczne.
Przegrzania spowoduje nieodwracalne szkody dla
jednostki!
Informações Importantes – Portuguese
• Se este equipamento é usado numa maneira não
especificada por Hoefer, Inc. que a protecção fornecida pelo equipamento pode ser comprometida.
• Este instrumento é projectado para uso de interior
de laboratório só.
• Só acessórios e partes aprovaram ou forneceu por
Hoefer, Inc. pode ser usada para operar, manter, e
servicing este produto.
• Só usa um estoque de poder que é CE marcou ou
segurança registrada por um nacionalmente reconhecido testando laboratório.
• A tampa de segurança deve estar em lugar antes
de ligar o estoque de poder leva a um estoque de
poder.
• Desliga todos controlos de estoque de poder e
desconecta os chumbos de poder antes de retirar a
tampa de segurança.
• Circulam só água ou 50/50 glicol de água/ethylene
pelo exchanger de calor se for assim equiparam.
Não ligue o exchanger de calor a uma torneira de
água nem qualquer fonte de refrigerante onde a
pressão de água é não regulado.
• Nunca introduz anticongelante nem qualquer
orgânico solvente em qualquer parte do instrumento. Orgânico solvente causará agressão
irreparável à unidade!
• Não opera com temperaturas de buffer acima do
máximo especificou especificações técnicas. Superaquecer causará agressão irreparável à unidade!
Información Importante – Spanish
• Si este equipo es utilizado en una manera no especificado por Hoefer, Inc. la protección proporcionado por el equipo puede ser dañada.
• Este instrumento es diseñado para el uso interior
del laboratorio sólo.
• Sólo accesorios y partes aprobaron o suministraron
• pv
por Hoefer, Inc. puede ser utilizado para operar,
para mantener, y para atender a este producto.
• Sólo utiliza una alimentación que es CE marcó o
la seguridad certificada por un nacionalmente
reconocido probando el laboratorio.
• La tapa de la seguridad debe estar en el lugar
antes de conectar la alimentación lleva a una
alimentación.
• Apaga todos controles de alimentación y desconecta los plomos del poder antes de quitar la tapa
de la seguridad.
• Circula sólo agua o 50/50 glicol de agua/etileno
por el intercambiador de calor si ése es el caso
equiparon. No conecte el intercambiador de calor a
un toque de la agua ni cualquier fuente del líquido
refrigerante donde la presión del agua está libre.
• Nunca introduce anticongelante ni algún solvente
orgánico en cualquier parte del instrumento. Los
solventes orgánicos causarán daño irreparable a
la unidad!
• No opera con temperaturas de búfer encima del
máximo especificó especificaciones técnicas. Recalentar causará daño irreparable a la unidad!
Viktig Information – Swedish
• om denna utrustning används i ett sätt som inte
har specificeras av Hoefer, Inc. skyddet tillhandahöll vid utrustningen kan skadas.
• Detta instrument formges för inomhuslaboratorium användning bara.
• Bara medhjälpare och delar godkände eller levererade vid Hoefer, Inc. kan användas för fungera,
underhålla, och servicing denna produkt.
• använder bara en kraft tillgång som är CE
markerade eller säkerhet intygade vid en nationellt
erkänd testande laboratorium.
• Säkerheten locket måste vara på platsen före
koppla kraften tillgången blyen till en kraft tillgång.
• Vänder sig alla kraft tillgång kontroller av och
kopplar bort kraften blyen före flytta säkerheten
locket.
• Cirkulerar bara vatten eller 50/50 vatten/ethylene
glycol genom värmen exchanger i så utrustad fall.
Inte kopplar värmen exchanger till en vatten kran
eller något kylmedel källa där vattnet trycket är
• pvi
unregulated.
• Inför aldrig kylvätska eller något organiska
lösningsmedel in i någon del av instrumentet.
Organiskt lösningsmedel ska orsaka irreparable
skada till enheten!
• Använd inte med buffert temperaturer över
det högsta angivna tekniska specifikationerna.
Överhettning skulle orsaka irreparabla skador på
enheten!
Waste Electrical and Electronic Equipment (WEEE)
English
This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal
waste and must be collected separately. Please contact an
authorized representative of the manufacturer for information
concerning the decommissioning of your equipment.
French
Ce symbole indique que les déchets relatifs à l’équipement
électrique et électronique ne doivent pas être jetés comme les
ordures ménagères non-triées et doivent être collectés séparément. Contactez un représentant agréé du fabricant pour obtenir des informations sur la mise au rebut de votre équipement.
German
Dieses Symbol kennzeichnet elektrische und elektronische
Geräte, die nicht mit dem gewöhnlichen, unsortierten Hausmüll entsorgt werden dürfen, sondern separat behandelt
werden müssen. Bitte nehmen Sie Kontakt mit einem autorisierten Beauftragten des Herstellers auf, um Informationen
hinsichtlich der Entsorgung Ihres Gerätes zu erhalten.
Italian
Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti
come rifiuti municipali indifferenziati e devono invece essere
raccolti separatamente. Per informazioni relative alle modalità
di smantellamento delle apparecchiature fuori uso, contattare
un rappresentante autorizzato del fabbricante.
Spanish
Este símbolo indica que el equipo eléctrico y electrónico no
debe tirarse con los desechos domésticos y debe tratarse por
separado. Contacte con el representante local del fabricante
para obtener más información sobre la forma de desechar el
equipo.
Swedish
Denna symbol anger att elektriska och elektroniska utrustningar inte får avyttras som osorterat hushållsavfall och
måste samlas in separat. Var god kontakta en auktoriserad
tillverkarrepresentant för information angående avyttring av
utrustningen.
• pvii
el electrophoresis unit G
function and description
The Hoefer SE600 series vertical slab gel
electrophoresis units (SE600, SE640 and SE660)
are intended for protein and nucleic acid electrophoresis under commonly used denaturing and
non-denaturing conditions. Up to 28 samples
can be compared on a single slab gel.
Applications include protein separations, nucleic
acid fractionation, and the second-dimension
separation of 2-D electrophoresis. First-dimension
separation of 2-D protein electrophoresis should
be performed on Immobilized pH Gradient Gels.
The focused strips are easily transferred to the
second-dimension slab gel for size separation.
The SE640 gel plates are 18 cm wide and 8 cm
in length. Up to four gels can be run at one time
if sandwiches are paired into “club sandwiches”.
• p1
Specifications
Gel plate size (w × h):
18 × 8 cm
Gel size:
14 × 8 cm
Maximum watt:
50 W
Maximum volt:
1000 V
Maximum ampere:
500 mA
Maximum temperature:
45 °C
Environmental operating conditions:
Indoor use: 4 – 40 °C
Humidity up to 80%
Altitude up to 2000 m
Installation category: II
Pollution degree: 2
Dimensions (w × h × d):32 × 22.5 × 14 cm
(12.5 × 8.75 × 5.5 in.)
Product certifications:EN61010-1,
UL61010A-1,
CSA C22.2 1010.1,
CE Certified
This declaration of conformity is only valid for the
instrument when it is:
• used in laboratory locations,
•u
sed as delivered from Hoefer, Inc. except for
alterations described in the user manual, and
• c onnected to other CE-labeled instruments or
products recommended or approved by Hoefer, Inc.
• p2
Fig 1. Main components of the
SE640 series (see Fig 4 for
caster components).
color-coded leads (2)
Included but not shown:
• Gel Seal compound, ¼ oz.
• Spacer-Mate spacer
positioning guide
• Glass plates (6)
• Wonder Wedge plate
separation tool
• Buffer dam
Complete unit also includes
spacers (4) and combs (2).
Required but not included:
• Magnetic stirrer
• Power supply with a minimum
rating of 300 V, 100 mA
(constant A or V)
Note: The ordering section on
page 38 lists all accessories
and replacement parts.
safety lid
upper buffer
chamber with
upper electrode
and lower
electrode fin
lower buffer
chamber
• p3
Unpacking and inventory
Note: Before using the first
time, disassemble the unit and
wash with a dilute solution of a
laboratory detergent and rinse
thoroughly first with water and
then with distilled water.
Unwrap all packages carefully and compare contents with the packing list, making sure all items
arrived. If any part is missing, contact your local
sales office. Inspect all components for damage
that may have occurred while the unit was in
transit. If any part appears damaged, contact the
carrier immediately. Be sure to keep all packing
material for damage claims or to use should it
become necessary to return the unit.
Lower buffer chamber
The lower buffer chamber is transparent acrylic,
which allows visual tracking of electrophoresis
progress. The chamber is chemically resistant to
common electrophoretic buffers but not to organic
solvents or strong acids and alkali. Temperatures
above 45 °C may cause the chamber to warp.
Upper buffer chamber
The upper buffer chamber is molded polysulfone, which is chemically resistant to common
electrophoretic buffers but not to organic solvents or strong acids and alkali. The upper electrode (cathode) runs along the center ridge, and
terminates at a banana plug. The lower electrode
(anode) runs along the edge of the electrode fin
on the underside, and terminates at a second
banana plug.
Safety lid
The banana plug on the upper buffer chamber
at the terminus of the cathode wire connects to
the black lead. The banana plug on the lower
electrode fin at the terminus of the anode wire
connects to the red lead. The 4-mm shrouded
color-coded leads plug into color-coded jacks in
the power supply. Always install the safety lid
before use!
• p4
Glass plates
The plates are 18 cm wide and 8 cm in length
Three sets of glass plates are included with each
unit. Notched divider plates, ordered separately,
pair two gel sandwiches to form a “club sandwich” so that up to four gels can be run at one
time.
Clamps
Clamps are used to secure the plates and spacers
together. The clamp pressure bar, adjusted with
screws, distributes pressure evenly.
Casting stand
The casting stand holds assembled gel sandwiches
upright for casting gels. Adjustable feet level the
caster. A laminated gasket in the bottom of each
casting cradle seals the bottom of the sandwich
when it is cammed into the stand.
Cams
Cams are used twice: first, to secure the assembled sandwich in the casting stand and, second, to
attach the sandwich to the upper buffer chamber.
Rubber gaskets
There are two sets of two gaskets: The solid
laminated gaskets fit into the bottom of the casting stand and form the seal for casting the gel.
The slotted gaskets fit under the upper buffer
chamber and form the seal between the upper
and lower chambers. The ridges on the upper
gasket align the gasket slot to maintain an open
channel between the top of the gel and the
buffer in the upper chamber.
• p5
Spacers
(May be ordered separately.) Spacers determine
the thickness of the gel and are available in three
thicknesses (0.75, 1.0, and 1.5 mm).
Spacer-Mate spacer positioning guide
Aligns spacers for sandwich assembly.
Combs
(May be ordered separately.) Combs are available in sizes that form 10, 12, 15, 20, or 28
wells, and are available in three thicknesses:
0.75, 1.0, and 1.5 mm. Preparative combs
include 1 or 2 reference wells in addition to the
single, large, preparative well.
All preparative combs, and 10, 12, 15 and
20-well combs form wells that are 25 mm deep.
The 28-well comb forms wells that are only 15
mm deep so that wells do not collapse when
the comb is removed. The sample volume held
by each well depends on the gel thickness, well
depth and the number of wells per comb. Table
1 on page 16 lists sample volumes of each well
for all combs.
Wonder Wedge plate separator tool
Used to disassemble gel sandwiches and to gauge
spacer and comb thickness.
• p6
Operating instructions
Gel casting and electrophoresis procedures
follow. Included are instructions for polyacrylamide gels (used with continuous or discontinuous buffer systems), and gradient gels.
The gels required for the SE640 must be selfcast. The Dual Gel Caster (included) holds two
gel sandwiches.
Prepare the gel sandwich
Glass plates, spacers, and clamp sets are sized
so that the assembled sandwich can be easily
aligned to create the seal required first to cast
the gel and then to run it. For best results, take
extra care to align all components when assembling sandwiches.
• p7
Construct the gel sandwich and insert into caster
1
both top and bottom
sandwich edges must
be flush with the
clamp guide ridges.
pressure
bar
Prepare the caster and clamps
Place the spirit level into the caster center and adjust
the leveling feet. Loosen all clamp screws and make
space for the sandwich by sliding the pressure plates
toward the screws.
2
Construct each gel sandwich
Fig 2. Sandwich assembly.
Inspect glass plates for nicks.
Use only unchipped plates to
prevent leaking.
Tip: Use the casting cradle
to hold the sandwich during
alignment. Remove the laminated gasket from the cradle
and, instead of setting the
sandwich upright on a flat
surface, set it into the casting cradle.
• p8
For each sandwich, choose two perfectly clean
unchipped glass plates and two spacers. Lay one
plate on a flat surface, lay the Spacer Mate spacer
positioning guide onto the plate (wide side at the top
of the plate), place a spacer along each edge, and lay
the second glass plate on top.
3
Secure the sandwich with clamps
Slide one clamp at a time along the sandwich sides.
Finger tighten one screw on each clamp, set the
sandwich upright on a flat surface, and loosen the
screw to align the stack. Take great care in aligning to
ensure a seal. Finger tighten all screws. Remove the
Spacer-Mate.
Club sandwich
A notched center divider plate (ordered separately)
pairs two sandwiches to double the number of gels
that can be cast and run.
glass plates
(at the outer sides
of the sandwich)
spacers
notched
center plate
Assemble a club sandwich in the same manner as
a regular sandwich, except before placing the top
glass plate, lay the divider plate and a second set
of spacers on the stack. Place the notch so that it
will be at the top of the gels. It is essential that the
spacers and plates align perfectly in order to create
a seal.
4
Remove the sandwich and inspect the bottom to make
sure that edges are aligned flush in order to ensure a
complete seal. Adjust if necessary.
Fig 3. Club sandwich assembly.
Side clamps will accommodate
two spacers up to 1.5 mm thick.
Optional: Apply a light film of Gel Seal compound only
on the bottom corner surfaces created by the spacers
and plates if your sandwiches continue to leak after
several attempts of alignment.
Note: Do not use silicone grease
or petroleum jelly to seal the
sandwich. These substances
are difficult to remove and ultimately cause artifacts.
• p9
5
Place the laminated gasket into the casting cradle
(See Fig 4) with the foam side down. Place the clamp
assembly in the casting cradle, screw side facing out.
6
Note: When turning the cams,
it is easier to keep the caster
balanced if you turn both toward
the center of the caster.
Insert a cam into the hole on each side of the casting
tray with the ridge (short end) pointing up. Seal the
gel sandwich against the casting gasket by turning
both cams as far as needed, usually 90° to 150°, up
to 180°. The cam action presses the plates down into
the gasket to seal the bottom of the sandwich. The
seal is complete once the glass edge appears darker
and nearly transparent against the gasket. Do not turn
the cam past this point.
clamp
Fig 4. Caster components and setup.
glass plate
spacer
gasket
(foam side down)
spirit level
casting cradles
leveling feet
• p10
cam
(install ridge
end up)
Acrylamide gels
1
Prepare the monomer solution and pour the gel
See Appendix A for SDS-PAGE recipes. Prepare the
required amount of monomer solution. De-aerate and
add the initiator (ammonium persulfate, APS) and
catalyst (TEMED) just prior to pouring the gel. Pipet
the solution into one corner of the sandwich, taking
care not to introduce any air bubbles. See below
for the appropriate solution level according to the
application.
No stacking gel (Continuous system)
Fill solution to just below the top of the upper plate
edge. If bubbles are trapped, remove with a pipette or
syringe. Introduce a comb (at a slight angle) into each
sandwich, taking care not to trap air bubbles under
the teeth.
Club sandwich Pipette the solution into both sandwiches, filling each
to the same level below the notched edge.
Stacking gel Fill solution to 3 – 4 cm below the top of the glass
plate. This height allows 1 cm of stacking gel below
the wells. Pour the gel and apply an overlay (see step
2). After the gel is set, prepare the stacking gel as
described below.
2-D electrophoresis (Discontinuous protein system)
Fill monomer solution to about 1 cm below the top
of the glass plate to allow 4 – 5 mm for the IPG strip
or tube gel and an agarose seal. (A stacking gel will
require extra space). Seal the IPG strip or tube gel in
place with agarose dissolved in running buffer. Take
care to avoid trapping any air bubbles between the
first- and second-dimension gels.
• p11
2
Overlay each gel with a thin layer of water-saturated
n-butanol, water, or diluted gel buffer to prevent
gel exposure to oxygen. Slowly deliver the overlay
solution from a glass syringe fitted with a 22-gauge
needle. Apply the solution near the spacer at the
side of the sandwich and allow it to flow across the
surface unaided.
3
Allow the gel to polymerize for a minimum of one hour.
Stacking gel preparation
Pour the stacking gel while the sandwich is still
in the gel caster. Stacking gel resolution is optimal when poured just before electrophoresis.
1
Remove the overlay by rinsing the top of the gel
several times with distilled water. Invert the caster
to drain. To ensure a seamless contact between the
resolving and stacking gels, remove residual liquid
by blotting one corner with a lint-free tissue.
2
Prepare the required amount of stacking gel monomer
solution, deaerate it, and add catalyst (APS) and
initiator (TEMED). Pour the stacking gel onto the
resolving gel with a disposable or Pasteur pipette to
a level about 2 mm from the top of the plate.
3
Introduce a comb (at a slight angle) into the sand­
wich, taking care not to trap air under the teeth. Allow
a minimum of one hour for the gel to polymerize.
• p12
Gradient gels
Both linear and exponential gradient gels can
be poured in the dual gel caster. We recommend
using a Hoefer SG Series Gradient Maker. Gradient gels are poured with a cannula from the
top of the dual gel caster (see Fig 5). A stacking
gel is then poured over the gradient gel.
Fig 5. Pouring a gradient gel.
The gel solution may be introduced into the gel sandwich
through a pipette tip at a rate that
maintains a continuous stream.
Pouring a linear gradient gel
1
Assemble sandwich(es) into the dual gel caster as
described on page 8.
2
Set up the monomer solution flow path
Run a length of Tygon™ tubing through a peristaltic
pump. Attach one end of the tubing to the gradient
maker outlet port and the other end to a 9 cm
cannula. (The OD of the cannula must be less than
the spacer thickness.) Place the cannula so that it
rests at the bottom of the sandwich, midway between
the spacers.
3
Optional: Adjust the higherpercentage acrylamide solution
to 15% (w/v) sucrose or
25% (v/v) glycerol to improve
layering.
Prepare the monomer solution
Calculate the volume of monomer solution needed.
Divide the total volume in half and prepare this
volume of both the higher- and lower-percentage
acrylamide solutions.
• p13
4
Pour the “light” solution into the reservoir chamber
(the chamber furthest from the inlet). Open the
stopcock long enough to displace air between the
chambers and then close. Pour the “heavy” solution
into the mixing chamber and place a stirring bar
into this chamber. Place the gradient maker onto a
magnetic stirrer and begin stirring at a rate that mixes
well but does not introduce bubbles into the solution.
5
Mix the gradient and pump the solution into the sandwich
While the solution is stirring, begin pumping from
the mixing chamber and open the stopcock to the
reservoir chamber. Raise the cannula as liquid enters
the sandwich, keeping the tip at the gel surface.
Prepare more gels as required.
6
Overlay each gel with a thin layer of water-saturated
n-butanol, water, or diluted gel buffer to prevent gel
exposure to oxygen. Slowly deliver the overlay solution
from a glass syringe fitted with a 22-gauge needle.
Apply the solution near the spacer at the side of
the sandwich and allow it to flow across the surface
unaided.
7
Allow the gels to polymerize for a minimum of one
hour. After polymerization, pour off the overlay and
rinse the gel surface several times with distilled water.
8
Prepare the stacking gel monomer solution, pour the
stacking gel and introduce a comb (at a slight angle)
into the sandwich, taking care not to trap air under
the teeth. Allow a minimum of one hour for the gel
to polymerize.
• p14
Sample preparation and loading
Note: With Coomassie™ Blue it
is possible to detect 1 µg of
protein in a single band. With
the more sensitive silver stains,
it is possible to detect as little
as 10 ng of protein.
The sample can be loaded either while the sandwich is in the caster or after the upper buffer
chamber is attached. When loading samples while
using divider plates, the samples must be loaded
without the upper buffer chamber in place.
The amount of sample loaded depends on the
thickness of the gel, the sensitivity of the detection method used, and the amount of sample
expected in each band. In a continuous buffer
system, the protein sample should be relatively
concentrated because no stacking gel is used.
In a discontinuous buffer system, the zone into
which each molecular species migrates is sharpened by the stacking gel so the sample need not
be as concentrated.
1
Prepare the wells
Remove the comb by gently rocking it side to side and
then lifting it straight up to avoid damaging the well
walls. Carefully rinse each well with distilled water to
remove unpolymerized acrylamide and then drain by
inverting the gel sandwich (or caster). Fill each well
with electrophoresis buffer.
2
Prepare the sample
Increase liquid sample density with 10% glycerol
or sucrose. Add a tracking dye such as phenol red,
bromophenol blue, or pyronin Y.
or SDS protein gels, use 2X treatment buffer to
F
denature both liquid and dry samples in a test tube.
To liquid protein solutions, add an equal volume of
2X buffer.
To dry protein samples, add equal volumes of 2X
sample buffer and high purity water to achieve the
desired concentration.
• p15
3
Heat the tube in boiling water for 90 seconds, then
allow to cool to room temperature. Treated samples
can be stored at -40 to -80 °C for future runs.
Note: Once the sample is in the
wells, take care to not jar the
sandwiches so that the samples
are not disturbed.
Heat membrane proteins to 60 °C for 20 minutes.
Store unused sample at 4 °C.
4
Underlay the sample into the wells using a fine-tipped
microsyringe or sample loading pipet tip.
Table 1. Sample volume for standard comb sizes
volume of sample (µl) per 1 mm depth
no. of comb thickness (mm) wells
0.75
1.0
1.5
• p16
10
6.2
8.3
12.4
12
5.8
7.7
11.5
15
4.3
5.7
8.6
20
3.1
4.1
6.2
28
2.1
2.7
4.1
1 prep/1 ref
90/4
121/6
183/9
1 prep/2 ref
85/4
113/6
171/9
Final assembly
Upper buffer chamber
1
Rinse both buffer chambers with water and distilled
water thoroughly before each use.
Clean away any gel adhering to the exterior of the
gel sandwiches.
2
If running only one gel:
Fig 6. Attaching gel sandwiches to
the upper buffer chamber.
If the assembly leaks, take it to a
sink and partially release the cams
to allow buffer to drain out of the
upper chamber. Disassemble, check
alignment of all sandwich components, and adjust if necessary.
A. R
emove cams from the lower
cam holes. Place the upper
chamber onto the sandwiches
and then insert the cams into
the upper cam holes, ridge
(short end) pointing down.
B. T
he final cam position (not
shown) must be vertical so that
the assembly fits into the lower
buffer chamber.
Note: Do not force the cams.
If encountering unusual resistance, disassemble and inspect
clamp and glass alignment
along the top of the sandwich.
Align and reinstall.
Block the second upper buffer chamber slot by
installing the acrylic buffer dam included with the
unit. Fit clamps onto the dam, taking care to align the
clamp ends and dam edges. Install the “dummy” gel,
screws facing out, in the second cradle in the dual gel
caster.
3
Attach the gel sandwich to the upper buffer chamber.
Turn the upper buffer chamber upside down and place
a slotted gasket into both sandwich holder recesses.
Both the slot in the gasket and the slot in the recess
must align. Both slotted gaskets must be used even
if running only one gel sandwich. Grooves along each
slot help keep the gasket in place. Additionally, a
small amount of Gel Seal can be applied at each end
of the gasket before install to help hold the gasket
against the upper buffer chamber.
Release the sandwiches from the caster by removing
all bottom cams (if present). Lower the upper buffer
chamber onto the gel sandwiches in the casting
stand. Install the cams, ridge pointing down, into
the buffer chamber cam holes. Cam the sandwich in
place by simultaneously turning one cam clockwise
and the other counterclockwise a full 180°.
• p17
4
Use a pipet to carefully fill each slot above the sample
wells with buffer in order to minimize disturbing the
samples. Then pour 100 ml of buffer into the chamber,
directing the buffer stream toward the side wall. Check
that no buffer is leaking around the gasket.
Lower buffer chamber
1
Place a magnetic spin bar into the lower buffer
chamber and place the unit on a magnetic stirrer.
Fill the lower chamber with a minimum of 2.1 liters
of buffer.
Optional: Prechill the buffer.
2
Fit the upper buffer chamber assembly into the lower
buffer chamber. Use a steady hand to avoid disturbing
the samples: Grasp the assembly in the casting stand
by the upper buffer chamber and carefully lower it
into the lower buffer chamber.
3
Inspect the installation and adjust the buffer levels.
Upper chamber. The electrode along the upper
chamber ridge must be submerged to a depth of
about 1 cm. This level requires 450 – 600 ml buffer:
just enough to cover the upper chamber ribs, but not
high enough to contact the banana plug.
Lower chamber. The lower buffer level chamber
requires a minimum of 2.1 liters and a maximum of
2.8 liters of buffer; enough to cover the wire on the
lower electrode fin, but maintaining a 1.5 cm clearance
from the underside of the upper buffer chamber.
4
Place the safety lid on the unit.
• p18
5
Plug the color-coded leads into the jacks of an
approved power supply. Plug the red lead into the
red output jack and the black lead into the black
output jack. In most systems, the red lead, which is
connected to the bottom electrode, is the anode (+),
and the black lead, connected to the top electrode,
is the cathode (–).
Important assembly notes:
•D
o not fill the upper or lower chamber above the
recommended levels illustrated in Fig 7. Remove
buffer in contact with the electrode posts.
•P
our buffer slowly and away from the slots in
the upper buffer chamber to avoid disturbing
the samples.
Fig 7. Buffer chamber levels.
upper chamber
buffer level
lower chamber
buffer level
• p19
Separating the sample
Electrophoresis parameters for discontinuous polyacrylamide gels
Note: All SE600 series models
use 18-cm wide plates. The
gel thickness determines the
cross section (and current
requirement). The length
of the plate determines the
running time.
Table 2: Laemmli buffer
system starting point guidelines
Gel thickness*
1.5 mm
Current per gel†
25 mA
constant
current
Starting voltage‡
80 – 90 V
Final voltage
220 – 400 V
*Thicker or thinner gels require proportionally more or less current. For
example, a 0.75 mm gel, which is half
as thick as a 1.5 mm gel, requires half
as much current, or 12.5 mA.
The current must be multiplied by
the number of gels. For instance, if
two club sandwiches are installed, the
four gels require four times as much
current. The current can be increased
for faster runs though overheating will
eventually become a problem, and it
can be decreased for slower overnight
runs.
†
‡
At 25 mA per gel.
Gels may be run at either constant current or
constant voltage settings. A constant current setting is traditionally used with a discontinuous
buffer system so that the rate of electrophoretic
migration remains unchanged throughout the
run. Under these conditions, voltage increases
as the run proceeds. A lower current setting is
recommended for higher resolution. The optimal
current level must be determined empirically;
the main factors that must be balanced include
the gel concentration and migration speed, and
the resulting Joule heating and band distortion.
Table 2 lists starting point guidelines and adjustments for gel thickness, number of gels, and
migration rate.
Current
Current acts on the total cross-section area of
all the gels because the gels are connected in
parallel in the electrical circuit. Thus the current
setting for one gel must be multiplied by the
number of gels of the same gel thickness that
are run simultaneously. For a gel 1.5 mm thick,
we suggest a starting current setting of 25 mA.
(Two 1.5 mm gels = 50 mA.)
Voltage
The starting voltage for a 1.5 mm slab gel connected to a power supply set to 25 mA is usually
80 to 90 V (using the SE600 with a Laemmli
discontinuous buffer system for SDS gels). The
final voltage can typically range from 220 to
400 V, depending on the length of the gel. (See
Table 2.)
• p20
Time
A run is complete when the tracking dye reaches
the bottom of the gel. A 1.5-mm thick Laemmli
SDS gel, run at 25 mA/gel without cooling, usually requires 2.5 hours.
Electrophoresis parameters for DNA/acrylamide gels
DNA gels are usually run at a constant voltage
setting, and since buffer systems are continuous,
both current and voltage readings remain constant throughout the run. Running conditions
are expressed in units of V/cm. Published running conditions vary widely, but voltages in the
range of 1 to 3 V/cm are common for overnight
runs.
Record each run
Caution! After initial monitoring,
do not leave the unit unattended
for more than 1 hour before
checking the progress of the
bands and the buffer level.
Keep a record of the current or voltage setting,
number and thickness of gels, buffer system, and
the starting and final current or voltage readings
for each run so that results can be compared.
Inconsistent results for the same system and settings indicate potential problems such as leaking
current, incorrect buffer concentrations, high salt
concentrations or inconsistent chemical quality.
Check band progress after 5 minutes, and again
after an hour, keeping an eye on the migration
rate of the tracking dye. The run is complete
when the tracking dye reaches the bottom of
the gel. Watch the buffer level and, if necessary,
replenish it as required to keep the top electrode
submerged. (A small volume of buffer may leak
past a nicked plate or gasket, or buffer may pass
through the gel.)
• p21
After electrophoresis
1
Once the tracking dye reaches the bottom of the
gel, turn off the power supply, disconnect the leads
and remove the safety lid. (Lift straight up to avoid
bending the banana plugs.)
2
Pull out the upper buffer chamber assembly. Pour the
buffer into a sink. Install the assembly in the dual
gel caster and then release the sandwiches by turning
and removing the cams.
3
Unscrew the clamps from the sandwiches and remove.
Gently loosen and then slide away both spacers. Use
the Hoefer Wonder Wedge plate separator tool to
separate the plates.
Note: Use only flexible plastic
prying tools to avoid chipping
the glass plates.
4
Carefully lift the glass plate with the gel attached.
Handle the gel with care to avoid damaging it. Invert
the plate and position the gel low over the staining
tray. Pry one corner of the gel away from the glass
and allow it to drop into the tray, or, if the gel is thick
enough to handle, lift it and place it into the tray. To
avoid splashing, add staining or fixative solution to the
tray after the gel is transferred.
5
Clean the unit as described in the next section.
• p22
Care and maintenance
Cleaning
Caution! Always unplug unit
from electrical supply before
cleaning or drying the unit.
Immediately after each use, rinse the upper and
lower buffer chambers with water and then
rinse thoroughly with distilled water. Handle
the upper buffer chamber with care to prevent
damage to the banana plugs and lower electrode
fin. Clean gaskets with mild detergent and rinse
with distilled water. Allow to air dry.
Clean glass plates and spacers with a dilute solution of a laboratory cleanser such as RBS-35,™
then rinse thoroughly with tap and distilled
water. Glass plates can also be treated with (but
not stored in) acid cleaning solutions.
• Do not autoclave or heat any part above 45 °C.
•D
o not use organic solvents, abrasives, strong
cleaning solutions, or strong acids or bases to clean
the chambers.
• Do not soak the laminated gasket.
• p23
Customer service information
Technical service and repair
IMPORTANT! Request a copy of
the Hoefer, Inc. “Health and
Safety Declaration” form before
returning the item. No items
can be accepted for servicing
or return unless this form is
properly completed.
Note: A Return Authorization
(RA) number must be obtained
from Hoefer, Inc, before returning any item to Hoefer, Inc.
• p24
Hoefer, Inc. offers complete technical support
for all of our products. If you have any questions about how to use this product, or would
like to arrange to repair it, please call or fax
your local Hoefer, Inc. representative.
Check the Hoefer, Inc. website at
www.hoeferinc.com for the distributor in your
area. Or contact us directly at:
Hoefer, Inc.
84 October Hill Road
Holliston, MA 01746
Toll Free: 1-800-227-4750
Phone: 1-508-893-8999
Fax: 1-508-893-0176
[email protected]
www.hoeferinc.com
Troubleshooting
problem
possible cause
remedy
Gel sandwich leaks while casting
Dirty or damaged
components
lates, spacers, and the gasket must be completely clean.
P
Wash if necessary.
Replace chipped plates (especially if chipped near the spacers).
Check the caster gasket for cuts or cracks and replace if
necessary.
Mis-aligned parts
Check plate and spacer alignment, realign if necessary.
Over-clamping
Turn cam only as far as necessary to create a seal (usually
90 –150°, but up to 180°).
n each spacer apply a light film of Gel Seal compound to
O
the bottom outside corner only. Do not use silicone grease.
Sample wells damaged or irregular
Air bubbles
emove air bubbles before inserting combs. Slide comb into
R
solution at an angle. If comb must be removed, add more
monomer solution before reinserting the comb.
Incomplete or
delayed
polymerization
Allow acrylamide gels to set for a minimum of 1 h.
Debris in wells
Rinse out unpolymerized gel with sample buffer.
Comb removal
Remove the comb at a slight angle and very slowly to prevent
damaging the gel.
Agarose gels: Lower the comb no more than 1 cm into the gel.
Incomplete gel polymerization
Chemicals
Use only recent stocks of the highest-quality reagents.
If the dry ammonium persulfate does not crackle when added
to water, replace with fresh stock.
Increase TEMED or APS concentration, or both.
pH
Solutions with extreme pH values (especially acidic) may not
polymerize.
Oxygen
emove oxygen from the gel environment: Degas the monoR
mer solution 5–10 min before pouring and then overlay the
gel surface with water-saturated n-butanol.
Temperature
Adjust the gel solution temperature to a minimum of 20 °C,
especially for low %T gels.
• p25
problem
possible cause
remedy
Upper buffer chamber leaks
Mis-aligned parts
heck that the glass plates, spacers, and clamps are aligned
C
and fit snugly into the upper chamber gasket.
Check that both gaskets are centered and that the positioning ridges fit inside the grooves.
Dirty or damaged
components
Check that the gasket is not damaged or pinched. Replace
if necessary. Check that the upper buffer chamber is not
warped from prior exposure to excessive heat.
Power supply detects current
leak
Electrical path to
outside ground/earth
Add more silicone grease to seal heat exchanger grommets.
Dye front curves up (smiles) at edges
Uneven heat
distribution
Fill the lower buffer chamber to the level appropriate for at
edges the run. (See Fig 7, page 19).
heck for leaks or cracks in the heat exchanger. Replace
C
worn grommets.
Use magnetic stirrer and stir bar to keep buffer well mixed.
Excessive heat
Circulate ext. coolant. Decrease the current or voltage setting.
Prechill the buffer. Run the gel in the cold room.
Protein streaks vertically
Unusually slow (or fast) run
Particulates in
sample
entrifuge or filter sample before loading to remove
C
particulates.
Overloading
Load less sample.
Degradation
Add protease inhibitor such as PMSF.
Current leakage
around gel
heck for leaks; all plates and spacers must be aligned and
C
free of grease and cracks.
If used, the buffer dam must be secure.
• p26
Sample or reagent
preparation
If the required pH of a solution is overshot, do not
back-titrate. Discard and prepare fresh buffer.
Check recipes, gel concentrations, and buffer dilution. (For
instance, do not use Tris-HCl instead of Tris for Laemmli
tank buffer.)
Decrease the salt concentration of samples.
Reagent quality
ispose of older acrylamide solutions and use only stock of
D
the highest quality. Use only freshly deionized urea.
oltage or current
V
settings
o increase or decrease the migration rate, adjust the voltage
T
or current by 25–50%.
problem
possible cause
remedy
Bands are skewed or
distorted
Incomplete gel
preparation and
polymerization
Degas the stacking-gel solution and avoid trapping air bubbles under the comb teeth.
Irregular interface
between stacking
and running gels
Overlay the running gel with water-saturated butanol before
polymerization begins, to avoid forming an uneven gel surface.
Sample preparation
Dialyze or desalt the sample.
Stained sample collects:
Near the buffer
front
Gel concentration
olecules are not sufficiently restricted by the resolving gel
M
pore size: increase the %T.
Degradation
roteins may be degraded by endogenous proteases: use proP
tease inhibitors during the isolation step.
Near the top of
the gel when
the buffer front
has reached the
bottom
Gel concentration
The gel pore size is too small: decrease the %T of the resolving (or stacking) gel.
Precipitation
The protein has precipitated. Heat the sample at a lower
temperature (70 °C or less) for 1–2 min.
At both top
and bottom of
the gel
Gel concentration
The molecular weight range of the sample requires an acrylamide concentration gradient to resolve the full range of
protein sizes.
Tracking dye Poor stacking
doesn’t sharpen into a concentrated
zone in the stacking gel
Reagent quality
Sample preparation
Pour a taller stacking gel. (For best results, allow a
stacking-gel height of 2.5 times the height of the sample in
the well.)
Dispose of outdated acrylamide solutions and use only the
highest grade of acrylamide.
hen preparing samples, avoid using solutions with high salt
W
concentrations.
• p27
problem
possible cause
remedy
Poor band resolution
Running
conditions
egin electrophoresis as soon as the sample is loaded to preB
vent low molecular weight species from diffusing.
Conduct the separation at a lower current or voltage setting
to reduce Joule heating.
Reagent quality
Use only the highest-quality reagents.
Poor stacking
Use only gels that were recently prepared.
dd a stacking gel or increase height of the stacking gel.
A
Prepare the resolving-gel surface by first rinsing it with
stacking-gel monomer before pouring the stacking gel to
ensure continuity between the gels.
Check pH values of the resolving- and stacking-gel solutions.
Do not back-titrate buffers.
Incomplete gel
polymerization
Allow gel to polymerize fully.
Sample
preparation
Store sample on ice before it is denatured.
Dialyze or desalt the sample.
eat samples in SDS sample buffer for no more than
H
1–2 min at 100 °C to improve dissociation of subunits.
Store on ice after heating.
Adjust the sample volume or concentration.
Add more mercaptoethanol or dithiothreitol; check sample
treatment.
dd protease inhibitors such as PMSF if necessary to preA
vent proteolytic degradation of sample.
Increase glycerol or sucrose to increase sample density.
tore samples to be frozen in aliquots to avoid repeated
S
freeze-thawing. Store at -40 to -80 °C.
• p28
Appendix A: Laemmli system gels
The Laemmli system is the most common electrophoresis protocol for SDS-denatured proteins.
The leading ion in this discontinuous buffer
system is chloride and the trailing ion is glycine.
Accordingly, the resolving gel and the stacking gel
contain Tris-Cl buffers (of different concentration
and pH), and the electrophoresis buffer contains
Tris-glycine. All buffers contain 0.1% SDS.
Polyacrylamide gel composition is indicated by two
different percentages:
% T = total acrylamide = g (acryl + bis) × 100
100 ml
% C = crosslinker = g (bis)
g (acryl + bis)
× 100
• p29
The total percent of acrylamide (% T) in the
resolving gel, which can range from 5 to 20%,
determines the pore size. Commonly, the amount
of crosslinker used (% C) is 2.6%. In the following example system, the resolving gel composition is 10% T, 2.6% C, which results in a
medium pore size. The stacking gel composition
is 4% T, 2.6% C. The % T in the stacking gel is
lower because a larger pore size is required.
Final concentrations
Separating gel Stacking gel Acrylamide conc. 10% T*, 2.6% C 4% T, 2.6% C
Tris-Cl 0.375 M 0.125 M
Electrophoresis buffer
Tris-Glycine 0.025 M Tris base
0.192 M glycine
pH 8.8 6.8 ~8.3
SDS 0.1% 0.1% 0.1%
Ammonium persulfate (APS) 0.05% w/v 0.05 – 0.1% w/v
TEMED† 0.05% v/v 0.05 – 0.1% v/v
*To achieve any other desired final concentration, adjust the acrylamide stock and water volumes. Volumes for different
concentrations are listed on page 34.
Tetramethylethylenediamine
†
• p30
Solutions
1. Acrylamide stock solution
(30.8% T 2.6% C Bis, 200 ml)
Note: Filter solutions 1– 4
through a 0.45 µm filter.
IMPORTANT! Refer to the material safety data sheet (MSDS)
accompanying each chemical
for detailed handling and safety
information.
Caution! Acrylamide is a neurotoxin. Always wear gloves while
handling in any form and wear
a mask while weighing the
powder. Never mouth pipette
the solution.
Acrylamide (FW 71.08)
30% w/v
60 g
Bis* (FW 154.2) 0.8% w/v
1.6 g
Deionized H2O to 200.0 ml
Store at 4 °C away from light.
*N,N’ Methylenebisacrylamide
2. 1.5 M TrisCl, pH 8.8
(4X Resolving gel buffer, 1 liter)
Tris (FW 121.1) 1.5 M 181.6 g
4 N HCl to pH 8.8
Deionized H2O to 1000 ml
3. 0.5 M TrisCl, pH 6.8
(4X Stacking gel buffer, 500 ml)
Tris (FW 121.1) 0.5 M 30.3 g
4 N HCl to pH 6.8
Deionized H2O to 500 ml
4. 10% SDS solution
(100 ml)
Sodium dodecylsulfate (SDS) (FW 288.4)
0.35 M
Deionized H2O 10.0 g
to 100 ml
5. 10% APS
(Initiator, 1 ml)
Ammonium persulfate
(APS) (FW 228.2)
0.44 mM
Deionized H2O 0.1 g
to 1.0 ml
Fresh APS “crackles” when water is added. If yours does not,
replace it with fresh stock. Prepare just prior to use.
• p31
6. 0.375 M TrisCl, 0.1% SDS, pH 8.8
(Resolving gel overlay, 100 ml)
1.5 M Tris-Cl, pH 8.8 (Soln. #2) 0.375 M 10% SDS (Soln. #4) 3.5 mM Deionized H2O 25.0 ml
1.0 ml
to 100.0 ml
—OR—
Water-saturated n-butanol
Shake n-butanol and deionized H2O in a separatory
funnel. Remove the aqueous (lower) phase. Repeat
this procedure several times. Use the upper phase.
—OR—
If an overlay interferes with the preferred protocol,
isolate the gel from atmospheric oxygen by placing a
blank comb or resolving gel former on the gel.
7. 2X Sample treatment buffer
(0.125 M TrisCl, 4% SDS, 20% glycerol,
2% 2-mercaptoethanol, pH 6.8, 10 ml)
0.5 M Tris-Cl, pH 6.8 (Soln. #3) 0.125 M 2.5 ml
10% SDS, 0.35 M (Soln. #4) 0.14 M 4.0 ml
Glycerol (FW 92.09) 20% v/v 2.0 ml
2-mercaptoethanol (FW 78.13) [–OR– Dithiothreitol (DTT) (FW 154.2) 2% v/v 0.2 mM 0.2 ml 0.31 g]
Bromophenol Blue (FW 691.9) 0.03 mM 0.2 mg
Deionized H2O to 10.0 ml
Divide into 1.0 ml aliquots and store at -40 °C to -80 °C.
—OR—
6X Sample treatment buffer
(0.35 M TrisCl, 10% SDS, 30% glycerol, 9.3% DTT,
pH 6.8, ~10 ml)
0.5 M Tris-Cl, pH 6.8 (Soln. #3) 0.35 M 7.0 ml
SDS (FW 288.4) 0.35 M 1.0 g
Glycerol (FW 92.09) 30% v/v 3.0 ml
DTT (FW 154.2) 0.6 M 0.93 g
Bromophenol Blue (FW 691.9)
0.175 mM 1.2 mg
Divide into 1.0 ml aliquots and store at -70 °C.
• p32
8. 0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3
(Electrophoresis buffer, 5.0 liters)
Tris (FW 121.1) 0.025 M 15.1 g
Glycine (FW 75.07) 0.192 M 72.0 g
SDS (FW 288.4) 3.5 mM Deionized H2O 5.0 g
to 5.0 liters
The pH of this buffer is approximately 8.3. Do not adjust pH.
Up to 20 liters can be prepared and stored for up to
2 months.
9. Coomassie stain solutions
Coomassie stain solution
(0.025% Coomassie Blue R-250,
40% Methanol, 7% Acetic acid, 2 liters)
Coomassie Blue R-250 (FW 826) Methanol (Stir until dissolved)
0.3 mM 0.5 g
40% v/v 800.0 ml
7% v/v 140.0 ml
Glacial acetic acid (99%) Deionized H2O to 2.0 liters
Destaining solution I
(40% methanol, 7% acetic acid, 1 liter)
Methanol 40% v/v Glacial acetic acid (99%) 7% v/v Deionized H2O 400.0 ml
70.0 ml
to 1.0 liter
Destaining solution II
(7% acetic acid, 5% methanol)
Methanol 5% v/v Glacial acetic acid (99%) 7% v/v Deionized H2O 50.0 ml
70.0 ml
to 1.0 liter
• p33
Gel recipes
The Laemmli gel recipes are for 30 ml of a single
concentration solution (enough for two 1.5 mm,
18 × 8 cm gels). Tabulated are ingredients and
volumes for relatively large pore gels (7.5 – 10% T
range) as well as smaller pore gels (12.5 – 15% T
range). A 4% stacking gel is common. The linear
gradient recipe is for 100 ml of solution. The
total volume needed depends on the number of
gels cast and the gel thickness; adjust as necessary.
All gels are crosslinked with 2.6% C.
Laemmli gel
(per 30 ml resolving gel solution, 5 ml stacking gel solution)
Separating gel
Stacking gel
7.5% 10% 12.5% 15% Acrylamide stock (Solution #1)
7.5 ml 10.0 ml 12.5 ml 15.0 ml 1.5 M TrisCl, pH 8.8 (Soln. #2) 7.5 ml 7.5 ml 7.5 ml 7.5 ml 0.5 M TrisCl, pH 6.8 (Soln. #3) 10% SDS (Solution #4) Deionized H2O 10% APS (Solution #5) —
0.3 ml 14.6 ml 150 µl —
0.3 ml 12.1 ml 150 µl —
0.3 ml 9.6 ml —
0.3 ml 7.1 ml 150 µl 150 µl 4%
0.67 ml
—
1.25 ml
0.05 ml
3.00 ml
25 µl
TEMED 10 µl 10 µl 10 µl 10 µl 2.5 µl
Final Volume 30.0 ml 30.0 ml 30.0 ml 30.0 ml
5.0 ml
• p34
For linear gradient gels, use equal volumes of
low % and high % acrylamide solutions in the
2 chambers of the gradient maker. Less APS is
added to extend polymerization time, and less
still is added to the higher %T solution to allow
polymerization to occur from the top down. In
our experience with the concentrations in the
10 – 20% gradient example below, gels can be
poured at a flow rate of 5 – 10 ml/min.
Linear gradient gel
(per 100 ml of solution)
Acrylamide stock (Solution #1) Sucrose 1.5 M TrisCl, pH 8.8 (Soln, #2)
10% SDS (Solution #4) Deionized H2O 10% T 20% T
33.30 ml 66.70 ml
—
15.00 g
25.00 ml 25.00 ml
1.00 ml 1.00 ml
a 100.00 ml a 100.00 ml
10% APS (Solution #5) 0.300 ml 0.060 ml
TEMED 0.036 ml 0.036 ml
• p35
Appendix B: Bibliography
General
Gallagher, S. R., and Smith, J. A., Electrophoretic separation
of proteins. In Current Protocols in Molecular Biology.
(Ausubel, F. A., eds.), OSC 10.2.1–10.2.21 (1991).
Hames, B. D., and Rickwood, D., Gel Electrophoresis of
Proteins: A Practical Approach: Second edition, City IRL
Press (1990).
Sambrook, J, Fritsch, E. F., and Maniatis, T., Standard Formaldehyde Protocol. Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY (1990).
Sasse, J., and Gallagher, S. R., Staining proteins in gels.
Current Protocols in Molecular Biology. (Ausubel, F. A.,
et al., eds.), OSC 10.6.1–10.6.8 (1991).
Non-denaturing gel systems
Reisfeld, R. A., et al., Acidic buffer system for resolution of
cationic proteins. Nature. 195, 281 (1962).
McLellan, T. Electrophoresis buffers for polyacrylamide gels at
various pH values. Anal. Biochem. 126, 94 (1982).
Hedrick, J. L. and Smith, A. J., Size and charge isomer separation and estimation of molecular weights of proteins by
discontinuous gel electrophoresis. Arch. Biochem. Biophys.
126, 155 (1968).
Denaturing gel systems
Laemmli, U. K. Cleavage of structural proteins during the
assembly of the head of bacteriophage T. Nature. 227,
680–685 (1970).
Matsudaira, P. T. and Burgess, D. R., SDS microslab linear
gradient polyacrylamide gel electrophoresis. Anal.
Biochem. 87, 386–396 (1978).
Schreier, M. H., Erni, B., and Staehelin, T., Initiation of
mammalian protein synthesis. I. Purification and characterization of seven initiation factors. J. Mol. Biol. Nov;
116(4):727–753 (1977).
Shapiro, A. L., and Maizel J. V. Jr., Molecular weight estimation of polypeptides by SDS-polyacrylamide gel electrophoresis: further data concerning resolving power and general
considerations. Anal. Biochem. Jun; 29(3):505–514 (1969).
Schaegger, H. and Von Jagow, G., Tricine-sodium dodecyl
sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal.
Biochem. 166, 368–379 (1987).
Weber, K., and Osborn, M., The reliability of molecular
weight determinators by dodecyl sulfate-polyacrylamide gel
electrophoresis. J. Biol. Chem. 224, 4406–4412 (1969).
• p36
Two-dimensional electrophoresis
Adams, L. D. and Gallagher, S. R., Two-Dimensional Gel Electrophoresis Using the O’Farrell System. Current Protocols
in Molecular Biology, (Ausubel, F. A., et al, eds.), OSC pp.
10.4.1–10.4.13 (1992).
Anderson, N. G., Anderson, N. L., and Tollaksen, S. L.,
Proteins of human urine. I. Concentration and analysis by
two-dimensional electrophoresis. Clin. Chem. Jul; 25(7):
1199–2210 (1979).
Anderson, Leigh and Anderson, Norman G., High resolution
two-dimensional electrophoresis of human plasma proteins.
Proc. Natl. Acad. Sci. USA. 74:5421–5425 (1977).
Anderson, L. Two-Dimensional Electrophoresis, Operation of
the ISO-DALT® System, Second Edition. Large Scale Biology
Press (1991).
Bravo, R., Schafer, R., Willecke, K., MacDonald-Bravo, H.,
Fey S. J., and Celis J. E., More than one-third of the discernible mouse polypeptides are not expressed in a Chinese
hamster-mouse embryo fibroblast hybrid that retains all
mouse chromosomes. Proc. Natl. Acad. Sci. USA. Apr;
79(7):2281–2285 (1982).
Hurkman, W. J., and Tanaka, C. K., Solubilization of Plant
Membrane Proteins for Analysis by Two-Dimensional Gel
Electrophoresis. Plant Physiology. 81:802–806 (1986).
Mets, L. J. and Bogorad, L. Two-dimensional polyacrylamide
gel electrophoresis: an improved method for ribosomal
proteins. Anal Biochem. Jan; 57(1):200–210 (1974).
O’Farrell, P. H., High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. May 25; 250(10):4007–
4021 (1975).
Bjellqvist, B., et al., Isoelectric focusing in immobilized pH
gradients: principle, methodology and some applications. J.
Biochem. Biophys. Methods 6, 317–339 (1982).
Görg, A, et al., The current state of two-dimensional
electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546 (1988).
Görg, A. Two-dimensional electrophoresis with immobilized
pH gradients: current state. Biochem. Soc. Trans. 21,
130–132 (1993).
Bjellqvist, B., et al., Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins. Electrophoresis 14, 1375–1378
(1993).
Blomberg, A., et al., Interlaboratory reproducibility of yeast
protein patterns analyzed by immobilized pH gradient
two-dimensional gel electrophoresis. Electrophoresis 16,
1935–1945 (1995).
• p37
Ordering information
product
quantity
code no.
SE640 Dual Vertical Unit, basic. Includes: 3 sets of glass plates, four 8 cm clamp
assemblies, 6 cams, dual gel casting stand with
leveling base and level, buffer dam, Spacer-Mate
alignment template and Wonder Wedge plate separation tool.
(Order 2 combs and 2 sets of 8 cm spacers separately.)
1
SE640
SE640 Dual Vertical Unit, complete. Includes: basic unit plus two 15-well combs and
2 sets of 8 cm spacers 1.5-mm thick.
1
SE640-15-1.5
Wonder Wedge gel plate separation tool 1
SE1514
Slotted silicone rubber gaskets for upper buffer chamber 2
SE6008B
Laminated silicone rubber gaskets for casting stand 2
SE6009
Replacement parts
Buffer dam 1
SE6432
Upper buffer chamber with electrode fin 1
SE6454
Lid with high voltage leads 1
SE6056
High-voltage safety lead set 1
SE6056-HV
Lower buffer chamber
1
SE6450
Replacement electrode fin for SE6454
1
SE6870
Banana plug, gold, with 2 washers
1
SE6067
Spirit level
1
SER11
Gel Seal, ¼ oz. tube
1
SE6070
• p38
product
quantity
code no.
1
SE6015
Gel Caster for 1 or 2 gels:
Dual Gel Caster, 1– 2 gels, 18 cm wide. Includes: 2 blank gaskets.
(One included with each SE640 unit)
Clamps and Cams
Replacement thumbscrews for clamps
12
SE6003U-2
Cams, black, for clamps with cam holes 4
SE6005L
Clamp assemblies, 8 cm
2
SE6403U
Glass plates
2
SE6402
Glass plates, low fluorescence
2
SE6402LF
Glass plate, club sandwich divider, notched
1
SE6402D
Glass Plates 18 × 8 cm
safety lid
with cables
SE6056
upper buffer
chamber with
electrode fin
SE6454
universal clamp
SE6403U
gasket
SE6009
basic caster
SE6015
lower buffer
chamber SE6450
spirit level
SER11
cam
SE6005L
• p39
Combs
number
of wells
a
thickness (mm)
width (mm)
quantity
code number
10
0.75
8.3
1
SE511-10-.75
10
1.00
8.3
1
SE511-10-1.0
10
1.50
8.3
1
SE511-10-1.5
12
0.75
7.6
1
SE511-12-.75
12
1.00
7.6
1
SE511-12-1.0
12
1.50
7.6
1
SE511-12-1.5
15
0.75
5.7
1
SE511-15-.75
15
1.00
5.7
1
SE511-15-1.0
15
1.50
5.7
1
SE511-15-1.5
20
0.75
4.1
1
SE511-20-.75
20
1.00
4.1
1
SE511-20-1.0
20
1.50
4.1
1
SE511-20-1.5
28a
0.75
2.7
1
SE511-28-.75
28a
1.00
2.7
1
SE511-28-1.0
28a
1.50
2.7
1
SE511-28-1.5
Comb depth 15 mm; all others 25 mm.
Preparative combs
These combs are 25 mm deep, adjustable to 10 or 15 mm.
no. of wells
prep/ref
thickness
(mm)
width (mm) prep/ref quantity
code number
1/1
0.75
121/6
1
SE511-R-.75
1/1
1
121/6
1
SE511-R-1.0
1/1
1.50
121/6
1
SE511-R-1.5
1/2
0.75
113/6
1
SE511-DR-.75
1/2
1
113/6
1
SE511-DR-1.0
1/2
1.50
113/6
1
SE511-DR-1.5
1
SE511-BKA
Adjustable comb back
Required to convert any 25-mm deep comb to 10 or 15 mm depth.
• p40
Spacers
thickness (mm)
length
width (mm)
quantity
2
2
2
2
2
2
SE6419-2-.75
SE6419-2-1.0
SE6419-2-1.5
Hoefer SE100 Plate Mate washing and storage unit
1
SE100
Hoefer TE62 Tank Transfer Unit
1
TE62
Hoefer TE70XP Semi-Dry Transfer Unit
1
TE70XP
0.75
1.00
1.50
8
8
8
code no.
Companion products
Hoefer reagents for gel casting and buffers
Acrylamide, MB grade
bis-Acrylamide, MB grade
1 kg
100 g
GR141-1
GR142-100
TEMED
25 g
GR151-25
Ammonium persulfate, ACS reagent grade
10 g
GR152-10
Tris-Glycine-SDS Buffer, 10X Solution, MB grade
1 L
GR149-1
Tris, reagent grade
1 kg
GR132-1
Glycine
1 kg
GR125-1
Sodium Dodecyl Sulfate (SDS)
Sodium Dodecyl Sulfate (SDS), 10% Solution
500 g
GR126-500
1 L
GR155-1
5 g
GR122-5
Hoefer reagents for sample loading and gel staining
Dithiothreitol (DTT), MB grade
EDTA, 0.5 M Solution, MB grade
Bromophenol Blue, sodium salt, ACS reagent grade
Glycerol, MB grade
Protein determination reagent, 500 assays
100 ml
10 g
1 L
500 ml
GR123-100
GR120-10
GR124-1
GR133-500
Coomassie Brilliant Blue G-250
25 g
GR134-25
Coomassie Brilliant Blue R-250
25 g
GR135-25
• p41
Hoefer, Inc. 84 October Hill Road
Holliston, MA 01746
Toll Free: 1-800-227-4750
Phone: 1-508-893-8999
Fax: 1-508-893-0176
E-mail: [email protected]
Web: www.hoeferinc.com
Hoefer is a registered trademark
of Hoefer, Inc.
Coomassie is a trademark of ICI plc.
RBS-35 is a trademark of Pierce
Chemical Co.
Tygon is a trademark of SaintGobain Performance Plastics.
© 2012 Hoefer, Inc. —
All rights reserved.
Printed in the USA
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