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Instruction Manual
Clari
arit-E® Vertical Electrophoresis Systems
Catalogue Numbers
EL2000, EL2004, EL2010, EL2002, EL2006, EL2008,
EL2018, EL2100, EL2110, EL2120, EL2184, EL2083,
EL2084, EL2390, EL2395, EL2318
Clari
arit-E® Complete Modular Systems
EL2190, EL2200, EL2250, EL2300
Clari
arit-E® Modular Vertical and 2-D Electrophoresis Systems
EL2310, EL2320, EL2026, EL2028, EL2330,
EL2096, EL2098, EL2340, EL2126, EL2128
Clari
arit-E® Modular Electroblotting Systems and Units
EL2400, EL2410, EL2450, EL2500, EL2402,
EL2034, EL2452, EL2114, EL2502, EL2134, EL2334
1
Contents:Page
1)
Safety Instructions
3
2)
Packing Lists
4
3)
Care and Maintenance
11
4)
System details
12
5)
Setting Up
13
6)
Gel Casting
13
7)
Gel Preparation
16
8)
Gel Selection
17
9)
Gel Pouring
19
10)
Sample Preparation and Loading
22
11)
Buffer Volume
23
12)
Gel Running
24
13)
Solutions
25
14)
References
26
15)
2-D Electrophoresis
27
16)
2-D tube gel pouring
27
17)
2-D Run conditions
29
18)
2-D Size determination phase
30
19)
Electroblotting
31
20)
Blot run conditions
32
21)
References
33
22)
Blotting Buffers
34
23)
DGGE instructions
35
14)
Combs
37
15)
Warranty
40
2
SAFETY PRECAUTION
WHEN USED CORRECTLY, THESE UNITS POSE NO HEALTH RISK.
HOWEVER, THESE UNITS CAN DELIVER DANGEROUS LEVELS OF ELECTRICITY
AND ARE TO BE OPERATED ONLY BY QUALIFIED PERSONNEL FOLLOWING THE
GUIDELINES LAID OUT IN THIS INSTRUCTION MANUAL.
ANYONE INTENDING TO USE THIS EQUIPMENT SHOULD READ THE COMPLETE
MANUAL THOROUGHLY.
THE UNIT MUST NEVER BE USED WITHOUT THE SAFETY LID CORRECTLY IN
POSITION.
THE UNIT SHOULD NOT BE USED IF THERE IS ANY SIGN OF DAMAGE TO THE
EXTERNAL TANK OR LID.
ACRYLAMIDE IS A POWERFUL NEUROTOXIN IN SOLUTION FORM.
POLYMERIZED GELS CAN CONTAIN SOME UNPOLYMERIZED SOLUTION AND
PROTECTIVE GLOVES AND CLOTHING MUST BE WORN.
THESE UNITS COMPLY WITH THE STATUTORY CE SAFETY DIRECTIVES:
73/23/EEC: LOW VOLTAGE DIRECTIVE: IEC 1010-1:1990 plus AMENDMENT 1:1992
EN 61010-1:1993/BS EN 61010-1:1993
3
PACKING LISTS:
EL2000, EL2004, EL2010, EL2002, EL2006, EL2008, EL2018
Units include tank, lid, internal module and electrodes and include the following
accessories:Glass Plates
Combs
Casting base
Cooling
Cables
Pack
EL2002
EL2004
EL2006
EL2000
EL2008
EL2040 – Notched, Pk/2
2 of EL2000-
EL2050 – Plain with bonded
12-1
1mm spacers, Pk/2
1mm thick, 12
EL2066 – Dummy Plate
sample
EL2040 – Notched, Pk/2
2 of EL2000-
EL2018
EL2050 – Plain with bonded
12-1
EL2020 – Mat
1mm spacers, Pk/2
1mm thick, 12
EL2066 – Dummy Plate
sample
EL2040
EL2714
EL2040
EL2714
EL2040
EL2714
SCREWS x 4
SCREWS x 4
EL2066– Dummy Plate
SCREWS x 4
EL2010
EL2018
EL2018
EL2020 - Mat
The packing lists should be referred to as soon as the units are received to
ensure that all components have been included. The unit should be checked for
damage when received. Please contact your supplier if there are any problems or
missing items.
4
EL2120, EL2100, EL2110, EL2184
Units include tank, lid, internal module and electrodes and include the following
accessories:Glass Plates
EL2120
Combs
EL2142 - Notched, Pk/2
2 of EL2100-24-1
EL2150 – Plain with bonded
1mm thick, 24
1mm spacers, Pk/2
sample
Casting
Cooling
base
Pack
Cables
EL2140
EL2714
EL2140
EL2714
EL2140
EL2714
EL2157 – Dummy Plate
EL2100
EL2142 - Notched, Pk/2
2 of EL2100-24-1
EL2110
EL2150 – Plain with bonded
1mm thick, 24
EL2090 - Mat
1mm spacers, Pk/2
sample
EL2157 – Dummy Plate
EL2184
EL2142 - Notched, Pk/2
2 of EL2100-24-1
EL2110
EL2150 – Plain with bonded
1mm thick, 24
EL2090 - Mat
1mm spacers, Pk/2
sample
Heater
EL2157 – Dummy Plate
Control
Unit
EL2186
EL2110
EL2110
EL2090 - Mat
The packing lists should be referred to as soon as the units are received to
ensure that all components have been included. The unit should be checked for
damage when received. Please contact your supplier if there are any problems or
missing items.
5
EL2083, EL2084
Units include tank, lid, internal module and electrodes and include the following
accessories:Glass Plates
Combs
Casting base
Cooling
Cables
Pack
EL2083
EL2084
EL2112 - Notched, Pk/2
2 of EL2100-
EL2121 – Plain with bonded
24-1
1mm spacers, Pk/2
1mm thick, 24
EL2156 – Dummy Plate
sample
EL2112 - Notched, Pk/2
2 of EL2100-
EL2088
EL2121 – Plain with bonded
24-1
El2090 - Mat
1mm spacers, Pk/2
1mm thick, 24
EL2156 – Dummy Plate
sample
EL2140
EL2714
EL2140
EL2714
EL2088
EL2088
El2090 - Mat
The packing lists should be referred to as soon as the units are received to ensure
that all components have been included. The unit should be checked for damage
when received. Please contact your supplier if there are any problems or missing
items.
6
EL2390, EL2395, EL2318
Units include tank, lid, internal module and electrodes and include the following
accessories:Glass Plates
Combs
Casting base
Cooling
Cables
Pack
EL2390
EL2395
EL2364 - Notched, Pk/2
2 of EL2390-1-
EL2382 – Plain with bonded
1.5
1.5mm spacers, Pk/2
1.5mm thick,
EL2386 – Dummy Plate
1 sample
EL2364 - Notched, Pk/2
2 of EL2390-1-
EL2318
EL2382 – Plain with bonded
1.5
EL2322 - Mat
1.5mm spacers, Pk/2
1.5mm thick,
EL2386 – Dummy Plate
1 sample
EL2342
EL2714
EL2342
EL2714
EL2318
EL2318
EL2322 - Mat
The packing lists should be referred to as soon as the units are received to ensure
that all components have been included. The unit should be checked for damage
when received. Please contact your supplier if there are any problems or missing
items.
7
EL2190, EL2200, EL2250, EL2300
Units include tank, lid, internal module and electrodes and include the following
accessories:-
EL2190
EL2200
Glass Plates
Combs
Casting base Cooling
Pack
Cables
EL2042 – Notched,
Pk/2
EL2050 – Plain with
bonded 1mm spacers,
Pk/2
EL2066 – Dummy
Plate
2 of EL2000-12-1
1mm thick, 12 sample
2 of EL2000-1-1.5
1.5mm thick, 1 sample
EL2018
EL2020 – Mat
EL2714
Spacers
EL2040
SCREWS x 4
2 of EL2074
Tube
Gel
Module
Capillary
Tubes
Blanking Plugs
Blotting
Module
Cassettes
Fibre
pads
EL2028
EL2030
EL2032
EL2034
3 of EL2036
2x
EL2038,
Pk/6
Glass Plates
Combs
Casting base Cooling
Pack
Cables
EL2142 - Notched,
Pk/2
EL2150 – Plain with
bonded 1mm spacers,
Pk/2
EL2157 – Dummy
Plate
2 of EL2100-24-1
1mm thick, 24 sample
2 of EL2100-1-1.5
1.5mm thick, 1 sample
EL2110
EL2090 – Mat
EL2140
EL2714
Tube
Gel
Module
Capillary
Tubes
Blanking Plugs
Blotting
Module
Cassettes
Fibre
pads
EL2128
EL2130
EL2032
EL2134
3 of EL2136
2x
EL2138,
Pk/6
EL2300
Spacers
2 of VS20S1.5
The packing lists should be referred to as soon as the units are received to ensure
that all components have been included. The unit should be checked for damage
when received. Please contact your supplier if there are any problems or missing
items.
8
Glass Plates
Combs
Casting base Cooling
Pack
EL2112 - Notched,
Pk/2
EL2121 – Plain with
bonded 1mm spacers,
Pk/2
EL2156 – Dummy
Plate
2 of EL2100-24-1
1mm thick, 24 sample
2 of EL2100-1-1.5
1.5mm thick, 1 sample
EL2110
EL2090 – Mat
Tube
Gel
Module
Capillary
Tubes
Blanking Plugs
EL2098
EL2030
EL2032
EL2250
Cables
EL2140
EL2714
Blotting
Module
Cassettes
Fibre
pads
EL2114
3 of EL2106
2x
EL2458,
Pk/6
Spacers
2 of EL2074
EL2310, EL2320, EL2330, EL2340
Units include tank, lid, internal module and electrodes and include the following
accessories:-
EL2310
EL2320
EL2026
EL2340
Glass Plates
Combs
Casting base
Cooling
Pack
Cables
EL2042 – Notched,
Pk/2
EL2050 – Plain with
bonded 1mm spacers,
Pk/2
EL2066 – Dummy Plate
2 of EL2000-12-1
1mm thick, 12 sample
2 of EL2000-1-1.5
1.5mm thick, 1 sample
EL2018
EL2020 - Mat
EL2040
EL2714
Tube Gel Module
Capillary
Tubes
Blanking
Plugs
EL2028
EL2030
EL2032
Glass Plates
Combs
Casting base Cooling
Pack
Cables
EL2142 - Notched,
Pk/2
EL2150 – Plain with
bonded 1mm spacers,
Pk/2
EL2157 – Dummy Plate
2 of EL2100-24-1
1mm thick, 24 sample
2 of EL2100-1-1.5
1.5mm thick, 1 sample
EL2110
EL2090 - Mat
EL2714
Tube Gel Module
Capillary
Spacers
2 of EL2074
SCREWS x 4
EL2140
Spacers
2 of EL2174
Blanking
9
EL2126
EL2330
EL2096
Tubes
Plugs
EL2128
EL2130
EL2032
Glass Plates
Combs
Casting base Cooling
Pack
Cables
EL2112 - Notched,
Pk/2
EL2121 – Plain with
bonded 1mm spacers,
Pk/2
EL2156 – Dummy Plate
2 of EL2100-24-1
1mm thick, 24 sample
2 of EL2100-1-1.5
1.5mm thick, 1 sample
EL2110
EL2090 - Mat
EL2714
Tube Gel Module
Capillary
Tubes
Blanking Plugs
EL2098
EL2030
EL2032
EL2140
Spacers
2 of EL2174
The packing lists should be referred to as soon as the units are received to ensure
that all components have been included. The unit should be checked for damage
when received. Please contact your supplier if there are any problems or missing
items.
EL2410, EL2400, EL2450, EL2500
Units include tank, lid, internal module and electrodes and include the following
accessories:-
EL2410
EL2400
EL2402
Glass Plates
Combs
Casting base Cooling
Pack
Cables
EL2042 – Notched,
Pk/2
EL2050 – Plain with
bonded 1mm spacers,
Pk/2
EL2066 – Dummy Plate
2 of EL2000-12-1
1mm thick, 12 sample
EL2018
EL2020 - Mat
EL2714
Blotting Module
Cassettes
Fibre pads
EL2034
3 of EL2036
2 of EL2038,
Pk/6
EL2040
SCREWS x 4
10
Glass Plates
Combs
Casting base Cooling
Pack
Cables
EL2500
EL2142 - Notched,
Pk/2
EL2150 – Plain with
bonded 1mm spacers,
Pk/2
EL2157 – Dummy Plate
2 of EL2100-24-1
1mm thick, 24 sample
EL2110
EL2090 - Mat
EL2714
Blotting Module
Cassettes
Fibre pads
EL2502
EL2134
3 of EL2136
2 of EL2138,
Pk/6
Glass Plates
Combs
Casting base Cooling
Pack
Cables
EL2450
EL2112 - Notched,
Pk/2
EL2121 – Plain with
bonded 1mm spacers,
Pk/2
EL2156 – Dummy Plate
2 of EL2100-24-1
1mm thick, 24 sample
EL2110
EL2090 - Mat
EL2714
Blotting Module
Cassettes
Fibre pads
EL2452
EL2114
3 of EL2106
2 of EL2458,
Pk/6
EL2140
EL2140
The packing lists should be referred to as soon as the units are received to ensure
that all components have been included. The unit should be checked for damage
when received. Please contact your supplier if there are any problems or missing
items.
11
Care and Maintenance:Cleaning Clarit-E® Units
Units are best cleaned using warm water and a mild detergent. Water at temperatures
above 60C can cause damage to the unit and components.
The tank should be thoroughly rinsed with warm water or distilled water to prevent build
up of salts but care should be taken not to damage the enclosed electrode and vigorous
cleaning is not necessary or advised.
Air drying is preferable before use.
The units should only be cleaned with the following:Warm water with a mild concentration of soap or other mild detergent.
Compatible detergents include dishwashing liquid, Hexane and Aliphatic hydrocarbons
The units should not be left to in detergents for more than 30 minutes.
The units should never come into contact with the following cleaning agents, these
will cause irreversible and accumulative damage:Acetone, Phenol, Chloroform, Carbon tetrachloride, Methanol, Ethanol, Isopropyl alcohol
Alkalis.
RNase Decontamination
This can be performed using the following protocol:Clean the units with a mild detergent as described above.
Wash with 3% hydrogen peroxide (H2O2) for 10 minutes.
Rinsed with 0.1% DEPC- (diethyl pyrocarbonate) treated distilled water,
Caution: DEPC is a suspected carcinogen. Always take the necessary precautions when
using. RNaseZAP™ (Ambion) can also be used. Please consult the instructions for use with
acrylic gel tanks.
12
Usage Guidance and restrictions:
• Maximum altitude 2,000m.
• Temperature range between 4C and 65C.
• Maximum relative humidity 80% for temperatures up to 31C decreasing linearly to 50%
relative humidity at 40C.
• Not for outdoor Use.
This apparatus is rated POLLUTION DEGREE 2 in accordance with IEC 664.
POLLUTION DEGREE 2, states that: “Normally only non-conductive pollution occurs.
Occasionally, however, a temporary conductivity caused by condensation must be
expected”.
Setting up the Clarit-E® Gel Tanks:-
Instructions for fitting Electrode Cables.
1. Note the position of the lid on the unit. This shows the correct polarity and the
correct orientation of the cables, black is negative and red positive.
2. Remove the lid from the unit. Note if the lid is not removed, fitting the cables may
result in un-tightening of the gold plug and damage to the electrode.
3. Screw the cables into the tapped holes as fully as possible so that there is no gap
between the lid and the leading edge of the cable fitting.
4. Refit the lid.
The unit is now ready to be used.
13
Vertical Gel Casting Using the Clarit-E® Gel Casting System:See page 12 for diagrams detailing the Mini vertical gel casting
procedure.
1. Clean a set of glass plates for each gel first with distilled water and then with 70 %
ethanol. One set of glass plates constitutes one notched glass plate and one plain glass
plate with bonded spacers. When using a triple glass plate sandwich, two notched glass
plates are required, one set of free spacers and a set of plain glass plates with bonded
spacers. The plain glass plate is positioned outermost, then a notched glass plate, free
spacers and second notched glass plate. Alternatively, accessory notch glass plates with
bonded spacers are available. All glass plates, modules and casting base accessories
must be completely dry during set – up. Wet components are more likely to missalign and cause leaks.
2. Assemble the glass plates so that the bottom of the glass plates and the spacers are
perfectly aligned. For triple plate sandwiches, the free spacers need to be perfectly
aligned which is best performed using a small spacer or comb to push the spacers apart.
Notched glass plates with bonded spacers do not need manual alignment. NOTE: The
glass plates with bonded spacers have an arrow in the top of the spacers which are
slightly longer than the glass plate to indicate the top.
3. The Slab Gel Insert contains pressure bars which impart even pressure onto the glass
plates and allow even screw pressure transfer onto the sealing edge of the glass plate,
ensuring complete sealing. Ensure that the pressure bars are adequately open for the
thickness of spacer used. The bar can be opened by loosening the screws or by sliding
the clamps. When using a triple glass plate sandwich, the pressure bars will need to be
in the completely open position.
14
4. Position the Slab Gel Insert on a flat surface. Do not at this stage insert the Slab Gel
Insert into the casting base.
5. Insert the glass plates into the Slab Gel Insert between the pressure bar and the blue
gasket and fully tighten the pressure bar screws in the order top then bottom. Fully
tighten the screw for the Mini vertical and the screws sequentially and in an even
manner for the maxi vertical in the order middle two, top then bottom, making sure not
to wobble the unit. When using the Slide Clamp Mini version, simply slide both gates
outwards until fully tightened. When only one gel is being run, the dummy plate must
be used in the second position and fully tightened. At this stage, check that the
bottom edges of the spacers and glass plates are perfectly aligned.
6. Position the Slab Gel Insert in the casting base such that the Cam pins have handles
pointing downwards and are located in the insert holes. The top of the GRM may need to
be pushed down very slightly to locate the cam pins.
With the cam pin handles facing directly downwards, turn the cam pins fully through 1800
or until the insert has tightened onto the silicone mat. It is best to turn the cams in
opposite directions to each other. Do not overturn as this will cause the glass plates
to push upwards and the assembly will be more likely to leak. The unit is now ready
for gel preparation and pouring
Always reverse the silicone mat after casting to avoid indentations from
persisting. Never leave the casting up-stand with glass plates tightened into the
casting base for long periods of time as this will also cause indentations in the
silicone mat.
15
The slide clamp version EL2000 also includes screws. This system can be used either
with the slide clamps or screws as preferred by the user. For those that prefer to use
the screws rather than clamps, the screws can be simply inserted into the screw
holes. The clamps can be removed by placing each clamp in the fully open position
and gently bending the clamp upwards from the slanted end. The holding pin will
then slowly release and the clamp can be removed.
VERTICAL GEL CASTING.
See step by step instructions on the following page
16
1) Put together bonded
spacer plain glass plate
with notched plate
2) Insert inside
pressure bar with
notched plate
innermost touching
the gasket and
module on a flat
surface away from
the casting base
A) SCREW VERSION OPTION
3A) Fully tighten
screws ensuring not
to wobble unit
4A) Insert into
casting base.
Push the cams
into the holes in
the insert. Turn
cams about 900
or until tight.
Do not over
tighten
B) SLIDING CLAMP VERSION OPTION
4B) Insert into
casting base.
Push the cams
into the holes in
the insert, turn
cams about 900
or until tight.
Do not over
tighten
3B) Fully Slide
Clamps tight
ensuring not to
wobble unit
5) Pour resolving gel
and allow to set. Then
stacking gel solution
and insert comb
6) Once set, transfer
to tank and fill inner
and outer chambers
with buffer
17
Gel Preparation:1. It is always advisable to work using stock solutions which allow added convenience and
save time when it comes to gel pouring. Pages 17 and 18 list stock solutions for SDS PAGE
gels which should be pre-made beforehand. For native gel formulae and running
conditions, please consult a laboratory manual. The protocol below is given for use of the
standard stock solutions advised. This should be adjusted if you are using different stock
solutions or gel formulas.
2. Table 1 below shows the total volume of gel solution required. In subsequent tables,
amounts of gel and solutions are given for two 1mm thick gels so adjustments are needed
for when running single or more than two gels and for 0.75, 1.5 or 2mm thick spacers.
Table 1.
Clarit-E® Mini – EL2000, EL2002,
EL2004, EL2006
Clarit-E® Maxi – EL2100, EL2120,
Clarit-E® MiniWide - EL2083, EL2084
Total Gel volume
Total Gel volume
for a 1mm thick
for a 1mm thick
gel.
gel.
For different thicknesses of gel, multiple the below amounts by the spacer
thickness. * multiply by 1.5 for Maxi Plus gels
Single – one gel,
one dummy plate
7.5ml
Double – two gels
Single – one gel,
Maxi 35ml*
one dummy plate
Miniwide 17.5ml
Double – two gels
Maxi 70ml*
15ml
Using a Triple Plate
sandwich – four
gels
Miniwide 35ml
Using a Triple Plate Maxi 140ml*
30ml
sandwich – four
gels
18
Miniwide 70ml
Gel Selection:Care should be taken when selecting the pore size of the gel to be used.
The pore size or % of gel determines the resolving ability given different sizes of protein.
See Table 2 below which details which percentage of gel to use to separate the sizes of
proteins indicated.
Table 2.
Acrylamide Percentage Separating Resolution
5%
60 - 220 KD
7.5 %
30 - 120 KD
10 %
20 - 75 KD
12%
17 – 65 KD
15 %
15 -45 KD
17.5%
12 – 30 KD
3. Prepare gel solutions as per tables below. These give the volumes of solutions from the
standard stock solutions. These should be gently mixed avoiding generation of bubbles
which will inhibit polymerization by removing free radicals.
19
Table 3: Preparation of the separating gel solution for two 10 x 10cm (Mini) gels using
1 mm spacers.
Solution
5%
7.5%
10 %
12%
15 %
17.5%
Distilled Water
8.7ml
7.5ml
6.3ml
5.25ml
3.75ml
2.5ml
30 % Stock
2.5ml
3.75ml
5ml
6ml
7.5ml
8.75ml
3.75ml
3.75ml
3.75ml
3.75ml
3.75ml
3.75ml
150µl
150µl
150µl
150µl
150µl
150µl
Acrylamide Solution
4 X Resolving Tris
Solution
10 % Ammonium
Persulphate
Table 4: Preparation of the separating gel solution for two 20 x 20cm (Maxi) gels using
1 mm spacers. Divide by two for 10 x 20cm (Miniwide) gels. Multiply by 1.5 for 30 x
22cm (Maxi plus) gels
Solution
5%
7.5%
Distilled Water
41ml
30 % Stock
10 %
12%
15 %
17.5%
35.25ml 29.6ml
24.7ml
17.6ml
11.7ml
11.7ml
17.6ml
23.5ml
28.2ml
35.25ml 41.1ml
17.6ml
17.6ml
17.6ml
17.6ml
17.6ml
17.6ml
700µl
700µl
700µl
700µl
700µl
700µl
Acrylamide
Solution
4 X Resolving Tris
Solution
10 % Ammonium
Persulphate
20
Gel Pouring:For gels with stacking layers:4. Insert the comb into the glass plates and mark a point on the glass plates 1cm below where
the comb teeth finish. This indicates where to add the resolving gel to.
5. Add 15µl of TEMED to the resolving gel solution for 10 x 10cm sized gels, 35 µl for 10 x
20cm, 70µl for 20 x 20cm and 105 µl for 30 x 22cm gels and mix well but avoid generating
air bubbles.
6. Fill the glass plates again avoiding generating any air bubbles. Filling must be performed
quickly before the TEMED causes the gel to become too viscous.
7. Overlay the gel extremely carefully with 1 ml of Isobutanol, Isopropanol or distilled water.
When using distilled water extra care must be taken to ensure there is no mixing with the
gel solution.
8. Let the resolving gel polymerize. Usually this takes around 15 minutes but this can vary
due to the freshness of the reagents used. If polymerization is taken a lot longer than this,
use fresher stock solutions or add more APS and TEMED.
9. Prepare the stacking gel using Table 5 on following page as a guide. Again stock solutions
are given on pages 17 and 18.
21
Table 5.
Solution
Mini
Mini wide
Maxi
Maxi Plus
Distilled Water
4.2ml
8.4ml
16.8ml
25.2ml
30 % Stock Acrylamide
0.65ml
1.3ml
2.6ml
3.9ml
1.6ml
3.2ml
6.4ml
9.6ml
67µl
134 µl
Solution
4 X Stacking Gel Tris
Solution
10 % Ammonium
268µl
Persulphate
10. Carefully mix the stacking gel solution, avoiding generating air bubbles.
11. Pour off the overlay liquid and rinse the gel with distilled water.
12. Add 6.7µl of TEMED to the stacking gel solution for Mini gels. For Miniwide gels add 13.4
µl, for Maxi gels add 26.8µl and 40.2 µl for Maxi Plus gels. Mix well. Use a Pasteur pipette
to fill the glass plates up to the top with stacking gel solution.
13. Carefully insert the comb making sure that no air bubbles get trapped under the ends of
the comb teeth as these will inhibit sample progression.
14. Allow the stacking gel polymerize for 30 minutes.
For gels without stacking layers:4. Add 15µl of TEMED to the resolving gel solution for Mini sized gels, 35 µl for Miniwide, 70µl
for Maxi and 105 µl for Maxi Plus gels and mix well but avoid generating air bubbles.
22
5. Fill the glass plates again avoiding generating any air bubbles. Filling must be performed
quickly before the TEMED causes the gel to become too viscous.
6. Carefully insert the comb making sure that no air bubbles get trapped under the ends of the
comb teeth as these will inhibit sample progression.
7. Let the gel polymerize. Usually this takes around 15 minutes but this can vary due to the
freshness of the reagents used. If polymerization is taken a lot longer than this, use fresher
stock solutions or add more APS and TEMED.
Preparation of denatured protein samples for loading:
The instructions given below are for denatured samples. For Native samples, please consult a
laboratory handbook.
1. Prepare the protein samples for loading. The volume of sample depends on the capacity of
the wells (See Comb specifications pages 22 and 23).
2. Using a 0.5 ml micro-centrifuge tube or other convenient receptacle, combine the protein
sample and 4x sample buffer. It is always advisable to use protein markers in one of the
end lanes to indicate sizes of bands. These should be prepared according to the
manufacturers instructions.
3. Heat the samples in a water bath or heating block for 2 minutes to denature the samples.
4. Centrifuge the samples in a micro-centrifuge for 20 seconds at 12,000 rpm. The protein
samples are now ready to load.
23
Loading the samples:
1. If desired, fit the cooling pack(s) into the end of the tank. These should be pre-frozen and
fitted with the longest side positioned sideways with the end(s) of the tank and pressed into
the recess. Or these can be fitted down the front of the tank.
NEVER FIT THESE UNDERNEATH THE MODULE IN THE BOTTOM OF THE TANK AS
THIS WILL PREVENT THE FLOW OF CURRENT THROUGH THE GEL AND CAUSE SLOW
RUNS AND OVER-HEATING.
Note one pack is supplied as standard. Additional packs can be purchased.
2. Transfer the Inner gel module containing cast gels into the main tank in the correct
orientation as indicated - +ve on the module aligned with
+ve on the tank, -ve on the
module aligned with –ve on the tank.
3. Fill the outer tank with 1x reservoir buffer. See page 22 for recommended running buffer
solution. Table 6. page 20 shows the volume of buffer required.
4. Load the samples into the wells using a pipette tip taking care not to damage the wells or
introduce any air bubbles.
5. Fill any unused wells with 1x sample buffer.
6. It is a good idea to note the orientation and order the samples were loaded in. This can be
done by noting which samples were loaded adjacent to each electrode.
24
Table 6.
Buffer Volume
Mini
Maxi,
Miniwide
Maxi plus
250ml
1.2Litres
500ml
1.8 Litres
Maximum – Inner tank is filled to above the wells.
1200ml
5.6 Litres
Outer Tank is filled to the maximum fill line. Cooling
2.8 Litres
8.4 Litres
Using the cooling packs – Inner tank is filled to
1000ml
4.6Litres
above the wells. Cooling packs are inserted behind
2.3 Litres
6.9 Litres
Minimum – Inner tank is filled to above the wells.
Outer Tank is filled to just flood the bottom of the
glass plates. Cooling potential is at a minimum which
may affect resolution.
is high offering good resolution of samples.
the gels. Outer Tank is filled to the maximum fill line.
Cooling is at a maximum.
25
Gel Running:
1. Fit the lid and connect to a power supply.
2. Consult Table 7, page 16 for details on recommended power supply voltage settings.
3. Turn the power supply off when the loading dye reaches the bottom of the gel, sooner if your
proteins are below 4Kd in size.
4. Remove the gel running module, first emptying the inner buffer into the main tank. Buffer
can be re-used but this may affect run quality if continued.
5. Unscrew the glass plates and gently pry apart the glass plates. The gel will usually stick to
one of the plates and can be removed by first soaking in buffer and then gently lifting with
a spatula.
6. The gel is now ready to be stained with Coomassie or silver stain or the proteins in the gel
can be transferred to a membrane by electroblotting for specific band identification and
further analysis.
Table 7.
Recommended Voltages and Resultant
Mini
Maxi,
Current for 1mm thick, 12% gels.
Miniwide
Maxi plus
90-225V
120-250V
20-45mA
20-45mA
90-225V
120-250V
40-90mA
40-90mA
90-225V
120-250V
60-135mA
60-135mA
90-225V
120-250V
80-180mA
80-180mA
One gel
Two gels
Three gels
Four gels
26
Stock Solutions for SDS PAGE gels:Stock 30% Acrylamide Gel Solution:30.0 g acrylamide
0.8 g methylene bisacrylamide
Distilled Water to 100ml
.
Stock 4 X Resolving Gel Tris (1.5 M Tris HCl pH8.8, 0.4 % SDS)
To 110ml Distilled Water add 36.4 g of Tris base
Add 8ml of 10 % SDS
Adjust pH to 8.8 with 1N HCl
Adjust the final volume to 200ml with Distilled Water.
.
Stock 4 X Stacking Tris (0.5 M Tris HCL pH6.8, 0.4 % SDS)
To 110ml Distilled Water add 12.12 g of Tris base
Add 8ml of 10 % SDS
Adjust pH to 6.8 with 1N HCl
Add Distilled Water to a final volume of 200ml
Stock 4 X Tris-glycine tank buffer - SDS
36 g Tris base
172.8 g glycine
Distilled Water to 3 L
27
1 x Tris-glycine tank buffer - SDS
750ml of 4 X Tris-glycine reservoir buffer - SDS
30ml of 10 % SDS
Distilled Water to 3L
10 % AP (ammonium persulphate solution)
0.1 g ammonium persulphate
1ml Distilled Water
TEMED
Stock 4 X Sample Buffer
4ml glycerol
2ml 2-mercaptoethanol
1.2 g SDS
5ml 4 X Stacking Tris
0.03 g Bromophenol blue
Aliquot into 1.5ml microcentrifuge tubes. Store at -20C.
28
References:-
1. Sambrook, Fritsch, and Maniatis, Molecular Cloning A Laboratory Manual, Second
Edition,
Cold Spring Harbor Laboratory Press, 1989.
2. Current Protocols in Molecular Biology, Greene Publishing Associates and WileyInterscience,1989.
29
1St Dimension Electrophoresis using the Clarit-E® Tube Gel
Module
Capillary Tube Gel Pouring:There are two methods which can be used for tube gel casting. Method 1 details casting by
injection, method 2 details casting by capillary action.
Method 1:- Filling By Injection
1. Place the appropriate number of capillary tubes into the Tube Gel Running module,
inserting these carefully from the top.
2. Seal the bottom ends of the tubes using NescoFilm.
3. Prepare the following solution. This will be enough to pour twenty 80mm Capillary Tubes
or ten 170mm Capillary Tubes. For Native IEF Gels, do not use Urea and NP-40 and use
18ml of distilled water instead of 16ml;
16ml Distilled Water (18ml for Native Gels)
2.4ml Glycerol
0.9ml 4-8 Resolyte or other commercially available 40% ampholyte solution
3.8ml Acrylamide/Bis solution
15µl TEMED
16.2g Urea (omit for Native Gels)
0.6ml NP-40 (omit for Native Gels)
This solution should be de-gassed prior to pouring.
When ready to pour, add 120µl of 10% w/v ammonium persulphate solution.
4. Using a Hamilton or similar syringe, insert the needle into the tube and carefully inject
the solution so that the tubes fill from the bottom. Keep filling to 1cm of the length of the
tubes. The tubes can be gently tapped to get rid of air bubbles.
30
5. Fill the remaining 1cm gap with water saturated isobutanol.
6. Leave to fully polymerise, which will normally take 1 – 2 hours.
7. After polymerisation, remove the water-saturated isobutanol. Tube gels can be used
immediately or stored wrapped in a damp paper towel and Nescofilm at 4oC. The Nescofilm
at the bottom of the tubes must be removed prior to electrophoresis.
Method 2:- Filling By Capillary Action
1. Place the appropriate amount of capillary tubes in a suitable outer receptacle such as a
15ml falcon tube.
2. The amount of acrylamide required depends on the size of the outer receptacle used. The
larger the outer receptacle used, the more acrylamide wastage so the following advised
volumes may need to be increased.
Prepare the following solution. This will be enough to pour twenty 80mm Capillary Tubes or
ten 170mm Capillary Tubes. For Native IEF Gels, do not use Urea and NP-40 and use 18ml
of distilled water instead of 16ml;
32ml Distilled Water (18ml for Native Gels)
4.8ml Glycerol
1.8ml 4-8 Resolyte or other commercially available 40% ampholyte solution
7.6ml Acrylamide/Bis solution
30µl TEMED
32.4g Urea (omit for Native Gels)
1.2ml NP40 (omit for Native Gels)
This solution should be de-gassed prior to pouring.
When ready to pour, add 240µl of 10% w/v ammonium persulphate solution.
31
3. Fill the falcon tube with 70% of the acrylamide solution. The capillary tubes will fill by
capillary action.
4. Allow the tubes to equilibrate for a few moments.
5. Check the height of the acrylamide in the tubes. If the tubes are full so that there is less
than a 1cm non-filled space at the top, remove some of the acrylamide solution from the
beaker until the height is 1 cm from the top. If there is a greater than 1cm space at the top,
add more acrylamide solution, so that the solution rises in the tubes until there is a 1cm
space at the top.
6. When the solution has reached to within 1cm of the top of the tube, stop adding the
acrylamide solution.
7. Fill the remaining 1cm gap with water saturated isobutanol.
8. Leave to fully polymerise, which will normally take 1 – 2 hours.
9. After polymerisation, remove the water-saturated isobutanol. Tube gels can be used
immediately or stored wrapped in a damp paper towel and Clingfilm at 4oC.
10. The tubes may contain a residual of acrylamide on the outside and may need cleaning
with distilled water before insertion into the tube gel insert.
32
1st Dimension (IEF) Phase Tube Gel Running
Buffer and run conditions will vary according to the type of ampholyte used. The following
conditions are given as guidelines only and apply when 4-8 Resolyte is the ampholyte used.
Other Ampholytes will require different buffer solutions. Please consult manufacturer’s
instructions.
1. Prepare ~ 500ml of 10mM H3PO4 Anode Buffer (1 litre for Mini, 2 litres for Maxi) and use
this to fill the bottom chamber of the unit so that the bottoms of the capillary tubes are
submerged. If less than 10 capillary tubes are to be run, block up the unused tube slots in
the internal running module with the blanking plugs provided. For high resolution
separations, we recommend filling the lower chamber completely with buffer and using a
pre-frozen cooling pack(s).
2. Place the Internal running Module into the unit and fill the upper buffer reservoir with
~100 mls of 20mM NaOH Cathode Buffer ( 200 mls for Mini, 400mls for Maxi) so that the
tops of the capillary tubes are submerged.
3. For the Prefocus, load the gels with 10µl of 1% ampholyte solution and run for 15
minutes at 200V, then for 30 minutes at 300 V and then finally 30 minutes at 400V. The
Prefocus stage is recommended as it helps set up the pH gradient.
4. Load the tubes with the samples. These should be dissolved in 1% ampholyte with 20%
glycerol.
5. Replace the safety lid firmly making sure that the electrical connectors form a good
contact.
6. Connect the electrophoresis apparatus to the power pack and connect the power pack to
the mains supply. Turn all settings to zero before turning on the mains supply.
33
7. Run at 400V for 3 hours and then 800V for 30 minutes. These conditions are for 8cm
tubes. 17cm tubes need to be run at 400V for 18 hours and then 800V for 1 hour.
8. At the end of the run, turn the power supply settings to zero, turn off the mains supply
and disconnect the power leads.
9. Remove the Internal Module and remove the tubes from their slots. The gels can be
extracted from the capillary tubes by: a) inserting a piece of wire with a small plug of cotton
wool on the end and using this as a piston to push the gel out, b) inserting a Gilson tip into
the end of the gel and gently squeezing the gel out with air or water. Whichever of these two
methods is used, the gels should be handled with care as they are fragile.
2-D, Size Determination Phase
1. To prepare the tube gel(s) for the 2-D, size-determining phase, equilibrate them by
soaking for 30 minutes in the running buffer to be used for the 2-D phase.
2. Remove the gel(s) from the running buffer pre-soak, and place each lengthways onto the
top of a pre-poured slab gel. The slab gel should be cast using a blank or 2-D comb. For
details on the casting of slab gels see the previous pages in this manual.
3. Hold the tube gel in place by pouring over it a low % agarose gel containing the tracker
dye.
4. Electrophorese as usual for Slab Gels until the tracker dye has advanced the required
distance down the gel.
5. The samples can be visualized using any of the standard staining methods or can be
blotted.
34
Protein Blotting using the Clarit-E® Electroblotter
Setting up the blot sandwich:
The most commonly used buffer solutions are given on page 34.
1. Each blot sandwich should be set up as follows:a. Cassette clamp -ve (black) side placed in a tray or other suitable surface.
b. Pre-soaked fibre pad. Note two can be used with thin gels.
c. Two pieces of thick filter paper, about 2 – 3 mm thick, pre-soaked in buffer.
d. Gel.
e. Transfer membrane. Usually this requires pre-soaking but consult the manufacturers
instructions for the type of membrane you are using. This should be smoothed so that no
air bubbles have been trapped.
f. Two pieces of thick filter paper, about 2 – 3 mm thick, pre-soaked in buffer.
g. Pre-soaked fibre pad. Note two can be used with thin gels.
h. Cassette clamp +ve (red) side slotted into the groove in the bottom of the black cassette.
2. Close the hinge carefully so as to not disturb the sandwich.
3. Fill the tank with buffer solution up to the maximum fill line indicated on the side of each
unit. See page 34 for recommended buffer solutions. Improved transfer can usually be
obtained by using chilled buffer.
Table 1. shows the volume of buffer required for each unit.
Buffer Volume
Mini
Miniwide
Maxi
Maxi Plus
One Cassette
1380ml
2800ml
5600ml
8400ml
Two Cassettes
1290ml
2620ml
5240ml
7860ml
Three Cassettes
1200ml
2440ml
4880ml
7320ml
35
Each Cooling pack will take the place or 100ml of buffer for Mini and 500ml of buffer for
Maxi, Miniwide and Maxi plus.
Blot Run Conditions:
1. Insert the cassettes into the slots in the module with the black side of each adjacent to the
negative electrode. It is a good idea to note the orientation and order the blot sandwiches
were loaded in. This can be done by noting which samples were loaded adjacent to each
electrode.
2. Use of a magnetic stirring bar and plate is recommended to mix the buffer to give
consistency of transfer. A 4mm diameter stirring bar should be placed underneath the
module, in the centre of the tank. The Cooling pack provided, pre-frozen, can be inserted at
the side or front of the tank for extended blots. Additional cooling packs can be purchased
as accessories to further aid cooling.
3. Insert the module, fit the lid and connect to a power supply.
4. Consult Table 2, Page 33 for details on recommended power supply voltage settings and blot
times. Please note voltages and current will vary according to the amount of cassettes, type
and temperature of buffer and thickness and percentage of gel. This will also affect quality
of transfer so time course of transfer should be performed for your particular samples and
conditions.
5. When the blot time is completed, turn the power supply off.
6. Remove the cassettes from the main tank. Buffer can be re-used but this may affect run
quality if continued.
7. Lift the hinge of each cassette and gently pry apart the blot sandwich and remove the
membrane from the gel.
8. The membrane is now ready to be probed.
36
Table 2. Recommended voltages and average resultant current.
Duration of Blot
Mini
Maxi
Miniwide
Maxi Plus
100V
100V
400mA
400mA
50V
50V
200mA
200mA
One Hours
Three Hours
References:1. Molecular Cloning A Laboratory Manual, Sambrook, Fritsch, and Maniatis, Second
Edition, Cold Spring Harbor Laboratory Press, 1989.
2. Current Protocols in Molecular Biology, Greene Publishing Associates and WileyInterscience,1989.
3. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: Procedure and some applications, Towbin, J., Staehelin, T., and Gordon, J.
(1979). Proc. Natl., Acad. Sci. USA, 76, 4350-4354.
4. Blotting Techniques Ch.1, 7.10, p. 85-97. In: Gel Electrophoresis of Proteins,
A Practical Approach, B.D.Hames and D.Rickwood, eds., IRL Press. (1990),
37
BUFFER SOLUTIONS:Towbin Buffer
25mM Tris,
192mM glycine,
20% methanol pH8.3,
Towbin Buffer SDS
25mM Tris
192mM glycine
20% methanol pH8.3
0.05-0.1% (w/v) SDS
Bjerrum and Schafer-Nielsen Buffer
48mM Tris
39mM glycine
20% methanol pH9.2
Dunn Buffer
10mM NaHCO3
3mM NaCO3
20% methanol pH9.9
Do not adjust the pH when making these buffers as this will cause blot over-heating.
The pH will vary according to the freshness of the reagents used.
38
EL2184 Instructions
SPECIFICATIONS:
Control Unit:
Size: 80 x 96 x 140mm ( H x W x D)
Mains Supply. 220 - 240V a/c. 50 - 60Hz
Mains Fuse. 5 Amp.
Input Fuse. 500mA, 240V Antisurge.
Output Voltage. 240 Volts.
Temperature Control Range. 0 - 200°C.
Controller Accuracy. +/- 2%.
Weight. 1.5Kg.
EL2184 Tank:
2x 250w Heating Elements
1x PT100 Thermocouple
Instructions:-
Installation and Operation of the EL2184 heating system
A. Installation
1. Connect the heating element plug into the socket at the rear of the control unit.
2. Connect the PT100 temperature sensing probe plug at the rear of the control unit.
3. Ensure the temperature dial is set to zero.
4. Connect the mains cable socket to the rear of the control unit then connect to an
external power source.
39
*CAUTION*
Do not attempt to apply temperature to the tank without any buffer present.
B. Using the Heating Controller
1. Fill the tank with buffer to the required level.
2. Ensure all the leads are connected in the correct positions as highlighted on the
instructions page on page 8.
3. Switch on the control unit to the rear of the unit and ensure that the red power light
illuminates.
4. Turn the dial on the front of the control unit to the required temperature. The small
red light will illuminate indicating the elements are switched on.
5. The heaters will remain on until the desired temperature has been reached, at which
point the unit will switch on and off to maintain that temperature.
6. To obtain an accurate temperature of the buffer, it may be advisable to attach a
temperature strip panel to the front of the tank.
C. Safety Considerations:
Should the sensor develop a fault or become disconnected the heaters will automatically
switch off so safeguarding against gel overheating.
To replace a fuse isolate on the control unit from the mains supply and open the fuse
holder with a screwdriver blade. The holder contains two fuses.
Always use the recommended fuse and NEVER replace it with one of a different rating.
40
Combs:–
MC Denotes Multi Channel Pipette compatible.
Mini Gel Tanks :0.5mm thickness
No. of Wells Catalogue
Number
1 Prep, 1 Marker EL2000-1-0.5
0.75mm thickness
Sample Vol. Catalogue
5mm thick gel Number
1mm thickness
Sample Vol. Catalogue
5mm thick gel Number
1.5mm thickness
Sample Vol. Catalogue
5mm thick gel Number
2mm thickness
Sample Vol. Catalogue
5mm thick gel Number
Sample Vol.
5mm thick gel
350µl
EL2000-1-0.75
500µl
EL2000-1-1
650µl
EL2000-1-1.5
1000µl
EL2000-1-2
1300µl
5
EL2000-5-0.5
40µl
EL2000-5-0.75
70µl
EL2000-5-1
100µl
EL2000-5-1.5
140µl
EL2000-5-2
200µl
8 MC
EL2000-8MC0.5
20µl
EL2000-8MC0.75
40µl
EL2000-8MC-1
60µl
EL2000-8MC1.5
80µl
EL2000-8MC-2
120µl
9
EL2000-9-0.5
20µl
EL2000-9-0.75
35µl
EL2000-9-1
50µl
EL2000-9-1.5
70µl
EL2000-9-2
100µl
10
EL2000-10-0.5
20µl
EL2000-10-0.75
30µl
EL2000-10-1
40µl
EL2000-10-1.5
30µl
EL2000-10-2
80µl
12
EL2000-12-0.5
15µl
EL2000-12-0.75
25µl
EL2000-12-1
35µl
EL2000-12-1.5
50µl
EL2000-12-2
70µl
16 MC
EL2000-16MC0.5
15µl
EL2000-16MC0.75
20µl
EL2000-16MC1
25µl
EL2000-16MC1.5
40µl
EL2000-16MC2
50µl
20
EL2000-20-0.5
10µl
EL2000-20-0.75
15µl
EL2000-20-1
20µl
EL2000-20-1.5
30µl
EL2000-20-2
40µl
Maxi and Miniwide Gel Tanks :0.75mm thickness
No. of Wells Catalogue
Number
1 Prep, 1
EL2100-1-0.75
Marker
1mm thickness
Sample Vol. Catalogue
5mm thick gel Number
1100µl
EL2100-1-1
1.5mm thickness
Sample Vol. Catalogue
5mm thick gel Number
1500µl
EL2100-1-1.5
2mm thickness
Sample Vol. Catalogue
5mm thick gel Number
2200µl
EL2100-1-2
Sample Vol.
5mm thick gel
3000µl
5
EL2100-5-0.75
160µl
EL2100-5-1
200µl
EL2100-5-1.5
320µl
EL2100-5-2
400µl
10
EL2100-10-0.75
80µl
EL2100-10-1
100µl
EL2100-10-1.5
160µl
EL2100-10-2
200µl
18 MC
EL2100-18MC0.75
40µl
EL2100-18MC1
50µl
EL2100-18MC1.5
80µl
EL2100-18MC2
100µl
24
EL2100-24-0.75
30µl
EL2100-24-1
40µl
EL2100-24-1.5
60µl
EL2100-24-2
80µl
30
EL2100-30-0.75
25µl
EL2100-30-1
35µl
EL2100-30-1.5
50µl
EL2100-30-2
70µl
36 MC
EL2100-36MC0.75
20µl
EL2100-36MC1
25µl
EL2100-36MC1.5
40µl
EL2100-36MC2
50µl
48
EL2100-48-0.75
15µl
EL2100-48-1
20µl
EL2100-48-1.5
30µl
EL2100-48-2
40µl
41
Maxi Plus Gel tanks :1mm thickness
No. of Wells
Catalogue Number
1 Prep, 1 Marker EL2390-1-1
1.5mm thickness
Sample Vol. Catalogue Number
5mm thick gel
Sample Vol.
5mm thick gel
2250µl
EL2390-1-1.5
3375µl
2
EL2390-2-1
1125µl
EL2390-2-1.5
1680µl
4
EL2390-4-1
550µl
EL2390-4-1.5
825µl
28 MC
EL2390-28MC-1
80µl
EL2390-28MC-1.5
120µl
56 MC
EL2390-56MC-1
40µl
EL2390-56MC-1.5
60µl
75 MC
EL2390-75MC-1
25µl
EL2390-75MC-1.5
37µl
Other combs available on request, please enquire
42
Notes
43
Warranty
The Alpha Laboratories Clarit-E® Electrophoresis units have a warranty against
manufacturing and material faults of thirty six months from date of customer receipt.
If any defects occur during this warranty period, Alpha Laboratories will repair or replace
the defective parts free of charge.
This warranty does not cover defects occurring by accident or misuse or defects caused by
improper operation.
Units where repair or modification has been performed by anyone other than Alpha
Laboratories or an appointed distributor or representative are no longer under warranty
from the time the unit was modified.
Units which have accessories or repaired parts not supplied by Alpha Laboratories or it’s
associated distributors have invalidated warranty.
Alpha Laboratories cannot repair or replace free of charge units where improper solutions
or chemicals have been used. For a list of these please see the Care and Maintenance
subsection.
If a problem does occur then please contact your supplier or Alpha Laboratories on:Alpha Laboratories Ltd.
40 Parham Drive,
Eastleigh,
Hampshire,
SO50 4NU
Tel: +44 (0)23 8048 3000
Fax: +44 (0)23 8064 3701
Email: [email protected]
44
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