Instructions for fitting Electrode Cables

Instruction Manual
Tank Sub
Electroblotting Systems
EBM10 EBM20
1
Contents:Page
1)
Safety Instructions
3
2)
Packing Lists
4
3)
System details
5
4)
Care and Maintenance
6
5)
Setting Up
7
6)
Preparation
8
7)
Run Conditions
9
8)
Solutions
10
9)
References
11
10)
Troubleshooting
12
11)
Warranty
17
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:
EBM10
Units include tank, lid, internal module and electrodes and include the following
accessories:Cassettes
Fibre Pads
Cooling
Cables
Pack
EBM10
5 of SB10C
2 of SB10F
Hinged
Fibre pads,
cassettes
Pk/6
VS10ICB
CSL-CAB
EBM20
Units include tank, lid, internal module and electrodes and include the following
accessories:Cassettes
Fibre Pads
Cooling
Cables
Pack
EBM20
5 of SB20C
2 of SB20F
Hinged
Fibre pads,
cassettes
Pk/6
VS20ICB
CSL-CAB
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
Usage Guidance and restrictions:
• Maximum altitude 2,000m.
• Temperature range between 4°C and 65°C.
• Maximum relative humidity 80% for temperatures up to 31OC decreasing
linearly to 50%
relative humidity at 40OC.
• 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”.
5
Care and Maintenance:Cleaning Tank Sub Blotting Units
Units are best cleaned using warm water and a mild detergent. Water at
temperatures above 600 C 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 preferably 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.
6
Setting up the Tank Sub Blotting Units: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.
7
Protein Blotting using the Tank Sub Electro-blotter:Setting up the blot sandwich:
The most commonly used buffer solutions are given on page 11.
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 11 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.
Table 1.
Buffer Volume
EBM10
EBM20
One Cassette
1500ml
6000ml
Two Cassettes
1410ml
5640ml
Three Cassettes
1320ml
5280ml
Four Cassettes
1230ml
4820ml
Five Cassettes
1140ml
4460ml
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Blot Run Conditions:
1. Insert the cassettes into the slots 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. This should be placed underneath the cassettes, in the
centre of the tank. Alternatively the cooling pack provided, pre-frozen, can be
inserted beneath the cassettes.
3. Fit the lid and connect to a power supply.
4. Consult Table 2 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.
Table 2. Recommended voltages and average resultant current.
Duration of Blot
One Hours
Three Hours
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EBM10
EBM20
100V
100V
400mA
400mA
50V
50V
200mA
200mA
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.
10
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
Wiley-Interscience,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),
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TROUBLESHOOTING
Poor protein transfer
Protein precipitating
in gel
Transfer apparatus assembled incorrectly and
proteins moving in the wrong direction
 Gel/membrane sandwich may be assembled in the
wrong order, or cassette inserted in wrong
orientation. Check polarity.
Western detection system not working or not sensitive
enough
 Include proper positive or negative control antigen.
Consult kit manual.
 Use protein markers with coloured reference bands
during PAGE (e.g. CSL-PPL or CSL-BBL).
 Stain gel with Coomassie, or stain membrane with
Ponceau S (CSL-PSS).
Transfer time too short – increase transfer time
Power setting too low
 Check current at beginning of run. Current may
be too low for a given voltage setting (see Table 2).
Increase current if necessary but do NOT exceed
2000mA.
 Buffer may be prepared improperly – prepare new
buffer and increase voltage.
Charge-to-mass ratio incorrect for native transfers.
 Proteins close to isoelectric point (pI). Change
buffer pH so that it is at least 2 pH unit higher or
lower than pI of protein of interest.
Defective or inappropriate power supply used.
 Check fuse of power supply. Ensure max. current
output of power supply is at least 2000mA (eg. CS3AMP).
Excessive methanol restricting transfer.
 Reduce methanol concentration to maximize
protein transfer from gel, but without reducing
concentration to the extent that it prevents binding
to nitrocellulose. Alternatively reduce methanol
concentration and switch to PVDF (CSLFPADSPVDF).
 Use SDS in transfer buffer (SDS can increase
transfer efficiency, but it can also reduce
nitrocellulose binding affinity and affect proteinantibody reactivity).
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 Remove alcohol from transfer buffer.
Swirls or missing
Poor gel-membrane contact. Air bubbles or excess
bands; diffuse transfers buffer remain between membrane and gel.
 Carefully remove air bubbles between gel and
membrane using a rolling pin
 Use more, or thicker, filter paper in gel-membrane
sandwich (e.g. CSL-BP1010, CSL-BP2020)
 Replace the fibre pads, as they degrade and remain
permanently compressed over time.
Membrane not fully wet or has dried out
 White spots on nitrocellulose membrane indicate
dry areas to which proteins will not bind. Ensure
membrane is completely immersed in transfer
buffer.
 If soaking does not occur immediately following
immersion in transfer buffer, heat distilled water to
just below boiling point and soak membrane until
entirely wet.
 If using PVDF, immerse membrane in methanol
before soaking in transfer buffer.
Problem with gel electrophoresis.
 Poor gel polymerization, inappropriate running
conditions, buffer contamination, excessive sample
application all contribute to poor quality gels and
transfers.
Gel cassette pattern
Contaminated fibre pads
transferred to blot
 Replace fibre pads or clean thoroughly.
Contaminated transfer buffer
 Replace buffer solutions.
 Use extra fibre pads and thicker blotting paper to
enhance compression
Poor binding to
Excessive methanol restricting transfer.
membrane  Ensure methanol concentration does not exceed
nitrocellulose
20% (v/v).
Proteins may be transferring through nitrocellulose.
 Use PVDF or smaller pore size (0.2µm)
nitrocellulose (e.g. CSL-RNC2)
 Overlay an extra piece of nitrocellulose over
membrane to determine if proteins are migrating
through the membrane directly in contact with the
gel.
Proteins <15kDa have reduced binding to 0.45µm
13
Poor binding to
membrane PVDF
Power is too high
Immune-specific
nitrocellulose or may be washed from membrane
during assays.
 Use PVDF or nylon membrane, which have higher
binding capacities.
 Use Tween-20 detergent in the wash and antibody
incubation steps. Reduce or eliminate the more
stringent washing steps.
SDS in transfer buffer reducing binding efficiency
 Reduce or eliminate SDS concentration
Membrane incompletely wet
 White spots indicate dry areas where protein will
not bind.
 If soaking does not occur immediately following
immersion in transfer buffer, heat distilled water to
just below boiling point and soak membrane until
entirely wet.
Membrane is not completely wet
 Because of hydrophobicity of PVDF, the
membrane must be soaked entirely in methanol
before equilibration in aqueous buffer
Proteins might be transferring through the
membrane
 Decrease voltage if transferring under high
intensity conditions
 Overlay an extra piece of PVDF over membrane to
determine if proteins are migrating through the
membrane directly in contact with the gel.
Membrane might have dried during handling
 Fully wet membranes have a grey translucent
appearance. White spots will form on the surface
if the membrane has been allowed to dry. As
proteins will not bind to dry spots, re-soak the
membrane in methanol and re-equilibrate in
transfer buffer
SDS in transfer buffer reducing binding efficiency
 Reduce or eliminate SDS concentration
Always check current at the start of the run, for the
current might be too high for a given voltage setting.
Improper buffer preparation can also result in high
conductivity and excessive power generation. The
current setting should not be allowed to exceed
2000mA.
Overall high background
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 Reduce antibody / protein sample concentration
Too low background
 Increase antibody concentration / protein sample
concentration
Consult manual included with antibody detection kit
Total protein detection Consult stain or detection kit manual.
detection
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Warranty
The Cleaver Scientific Ltd. (CSL) Electrophoblotting units have a warranty
against manufacturing and material faults of twelve months from date of
customer receipt.
If any defects occur during this warranty period, CSL 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
CSL 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 CSL or it’s
associated distributors have invalidated warranty.
CSL 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 CSL on:Cleaver Scientific Ltd.
Unit 4 Triton Park
Swift Valley
Brownsover Road
Rugby
CV21 1SG
Tel: +44 (0)1788 565300
Fax: +44 (0)1788 552822
Email: info@cleaverscientific.com
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