Labnet International Vertical Gel Electrophoresis Systems

Labnet International Vertical Gel Electrophoresis Systems
Labnet International Vertical Gel Electrophoresis
Systems
Catalog Number
Mini PAGE System
E2010-P
Mini PAGE
Electroblotting System
E2010-PB
Mini PAGE
Tube Gel System
E2010-P2
1
Contents:
Page
1)
Safety Precautions
3
2)
Packing Lists
4
3)
Care and Maintenance
5
4)
Setting Up
5
5)
Gel Casting
6
6)
Gel Preparation
7
7)
Gel Selection
7
8)
Gel Pouring
8
9)
Sample Preparation and Loading
9
10)
Gel Running
10
11)
Blotting Insert Setup
12
12)
Blot Running
12
13)
2-D Gel Insert Setup
14
14)
2-D Gel Running
15
15)
Appendix
17
16)
Warranty
19
17)
Accessories List
20
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 current to the unit, provided from external power supply, enters through the lid
assembly, providing a safety interlock to the user. When the lid is removed, the
current to the unit is broken. DO NOT attempt to use the unit without the safety lid
correctly positioned. Always turn the power supply off prior to removing the lid.
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, SAFETY GLASSES 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:
Vertical Mini
Units include tank, lid, internal module and electrodes and include the following accessories:Glass Plates
Combs
Casting
Cooling
base
Pack
Included
Mini PAGE
Glass Plates, Notched, Pk/2
2, 1mm thick,
Base with
System
Glass Plates, Plain with
12 sample
Silicone Mat
bonded 1mm spacers, Pk/2
combs
Cables
Red
Black
Buffer dam
Vertical Mini with Western Blotting Insert
Units include tank, lid, internal module, electrodes, Blotting module and include the following
accessories:Glass Plates
Combs
Casting
Cooling
base
Pack
Included
Mini PAGE
Glass Plates, Notched, Pk/2
2, 1mm thick,
Base with
system with
Glass Plates, Plain with
12 sample
Silicone Mat
Electroblotting
bonded 1mm spacers, Pk/2
combs
system
Buffer Dam
Electroblotting
3 Cassettes, Pack of 6 Fiber pads
Cables
Red
Black
module
Vertical Mini with 2-D Tube Gel Insert
Units include tank, lid, internal module, electrodes and capillary support frame and include the
following accessories:Glass Plates
Combs
Casting
Cooling
base
Pack
Included
Mini PAGE
Glass Plates, Notched, Pk/2
2, 1mm thick,
Base with
with 2-D Tube
Glass Plates, Plain with
12 sample
Silicone Mat
Gel module
bonded 1mm spacers, Pk/2
combs
Cables
Red
Black
Buffer Dam
2-D Tube Gel
10 Capillary Tubes, 10 Blanking plugs
module
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. If
damaged, contact carrier and save the box for inspection. Please contact VWR if there are any
problems or missing items.
4
Usage Guidance and restrictions:
• Maximum altitude 2,000m.
• Temperature range between 4°C and 60°C.
• Maximum relative humidity 80% for temperatures up to 31°C decreasing linearly to 50% relative
humidity at 40°C.
• 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”.
Care and Maintenance:Cleaning Vertical Units
Units are best cleaned using warm water and a mild detergent. Water at temperatures above 60° C
can cause damage to the unit and components. The tank should be thoroughly rinsed with warm
water and distilled water to prevent build up of salts but care should be taken not to damage the
enclosed electrode. Vigorous cleaning is not necessary or advised. Air drying is recommended
before use.
The units should never come into contact with the following cleaning agents, these will cause
irreversible and cumulative 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.
Rinse with 0.1% DEPC- (diethyl pyrocarbonate) treated distilled water,
Caution: DEPC is a suspected carcinogen. Always wear gloves and safety glasses.
RNaseZAP™ (Ambion) can also be used. Please consult the instructions for use with acrylic gel
tanks.
Setting up the Vertical Mini Gel Tanks:-
Instructions for installing Electrical Leads.
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.
5
2. Remove the lid from the unit. Note if the lid is not removed, fitting the cables may result in untightening of the gold plug and damage to the electrode.
3. Screw the cables into the tapped holes in the lid 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 for use.
Vertical Gel Casting Using the Mini PAGE Gel Casting System: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, one set of free
spacers and a set of plain glass plates with bonded spacer. The plain glass plate is positioned
outermost, then a notched glass plate, free spacers and second notched glass plate. Alternatively,
accessory notched glass plates with bonded spacers are available. All glass plates, modules
and casting base accessories must be completely dry for setup. Wet components are
more likely to misalign 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 spacers bonded to the glass plates
are slightly longer than the plates and marked with an arrow to indicate the top.
3. The Mini PAGE Gel insert contains pressure bars which impart even pressure onto the edge of
the glass plate. Ensure that the pressure bars are adequately open for the thickness of spacer
used. The bar can be opened by loosening the screws. When using a triple glass plate sandwich,
the pressure bars will need to be in the completely open position.
4. Position the Mini PAGE Gel module on a flat surface. Do not insert the Mini PAGE Gel module
into the casting base at this point.
5. Place the glass plate/spacer assembly into the Vertical Gel Insert between the pressure bar and
the blue gasket. Check that the bottoms of the glass plates are touching the bench and fully
tighten the pressure bar screws in the order top to bottom. Care must be taken not to over-tighten
the screws or breakage will occur. When only one gel is being run, the buffer dam must be used
in the second position and fully tightened. NOTE: Be sure that the glass plates and the
bottom of the spacers are evenly aligned.
6. Open the cam handles on the casting stand and position the cams so they face downwards.
Position the Mini PAGE Gel insert on the gasket of the casting stand so the key holes are facing
the cams. The top of the Mini PAGE Gel insert will need to be pushed down very slightly to insert
the cams.
6
7. With the cam handles facing directly downwards, rotate the cams in the opposite direction 1800
or until the insert has tightened down onto the gasket. Do not overturn as this will cause the
glass plates to push upwards and the assembly will be more likely to leak.
NOTE: Always reverse the gasket after casting to avoid indentations. Do not leave glass plates
tightened into the casting stand for long periods of time as this can cause damage the
silicone mat. The unit is now ready for gel preparation.
Gel Preparation:1. Stock solutions for SDS PAGE gels should be prepared prior to use and chilled. For native gel
formulas and running conditions, please consult the appendix. The protocol below is given for use
of the standard stock solutions.
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.
Table 1.
Vertical Mini PAGE
Total Gel volume for a 1mm thick gel.
For different thicknesses of gel, multiple the below amounts by the spacer thickness.
Single – one gel, one dummy plate
7.5 mL
Double – two gels
15 mL
Using a Triple Plate sandwich – four gels
30 mL
Gel Selection:Care should be taken when selecting the pore size of the gel to be used. These formulas are for Trisglycine-SDS gels
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
5%
7.5 %
10 %
12%
15 %
17.5%
Separating Resolution
60 - 220 KD
45 - 120 KD
25 - 75 KD
14.4 – 65 KD
6.5 -45 KD
5.5 – 30 KD
7
3. Using the stock solution provided in the appendix prepare gel solutions as per tables below. First
mix the ddi Water, 30% Acrylamide solution and the 4x TRIS-SDS solutions. After mixing, degas
for 5 minutes to remove free oxygen (which will inhibit polymerization).
4. To the above solution add the ammonium persulfate and TEMED and mix gently to avoid air
bubbles.
Table 3: Preparation of the separating gel solution for two 10 x 10cm gels using 1 mm spacers.
Solution
5%
7.50%
10%
12%
15%
17.5%
Distilled Water
8.7 mL
7. 5mL
6.3 mL
5.25 mL
3.7 5mL
2.5 mL
30 % Stock
Acrylamide
Solution
2.5 mL
3.75 mL
5 mL
6 mL
7.5 mL
8.7 5mL
4 X Tris-SDS
Solution pH 8.8
3.75 mL
3.75 mL
3.75 mL
3.75 mL
3.75 mL
3.7 5mL
10 % Ammonium
Persulphate
150 µl
150 µl
150 µl
150 µl
150 µl
150 µl
TEMED
15 µl
15 µl
15 µl
15 µl
15 µl
15 µl
Gel Pouring:For discontinuous gels:1. Insert the comb between the glass plates and mark a point on the glass plates 1cm below the
bottom of the teeth. This is the level for the resolving gel.
2. Fill the glass plates to the line and avoid generating any air bubbles. Filling must be performed
quickly before the TEMED causes the gel to become too viscous.
3. Carefully, overlay the gel with 1 ml of 1% Isobutanol, Isopropanol or distilled water. When using
distilled water, extra care must be taken to ensure there is no mixing with the gel solution.
4. Allow the resolving gel to polymerize. This usually takes 15 to 3 minutes. If polymerization time
is excessive, use a fresh stock of APS.
5. Prepare the stacking gel using Table 5 below as a guide. (See appendix for stock solutions.)
Table 5.
Solution
Distilled Water
Vertical Mini
4.2 mL
30 % Stock Acrylamide
Solution
4 X Stacking Gel Tris-SDS
Solution pH 6.6
0.65 mL
10 % Ammonium Persulphate
67µl
TEMED
6.7 µl
1.6 mL
8
6. Carefully mix the ddi water, acrylamide and Tris-SDS solution, degas for 5 minutes.
7. Add the ammonium persulfate and the TEMED and mix.
8. Pour off the overlay liquid and rinse the gel with distilled water.
9. Use a Pasteur pipette to fill the glass plates up to the top with the stacking gel solution.
10. Carefully insert the comb making sure that no air bubbles are trapped under the ends teeth as
these will inhibit sample progression.
11. Allow the stacking gel polymerize for 30 minutes.
For continuous gels:1. Follow the instructions for mixing the acrylamide solution.
2. Fill the glass plates to 1 cm below top of notched plate, again avoiding generating any air bubbles.
Filling must be performed quickly before the TEMED causes the gel to become too viscous.
3. Carefully insert the comb making sure that no air bubbles are trapped under the teeth as these
will inhibit sample progression.
4. Let the gel polymerize. This usually takes 15 to 30 minutes but can vary. If polymerization takes
longer than expected, use a fresh stock solution of APS.
Preparation of denatured protein samples for loading:
The instructions given below are for denatured samples. For Native samples, please see appendix.
1. Prepare the protein samples for loading. The volume of sample depends on the capacity of the
wells. See well chart in the Appendix.
2. Using a 0.5 mL microcentrifuge tube or other convenient receptacle, combine equal volumes of
the loading dye, protein sample and 2X sample buffer. It is always advisable to use protein
ladders 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.
NOTE heating should be done under a fume hood.
4. Centrifuge the samples in a microcentrifuge for 20 seconds at 12,000 rpm. The protein samples
are now ready to load.
Loading the samples:
9
1. If desired, fit the previously frozen cooling pack(s) into the tank. The longest side of the ice pack
should be positioned horizontal with the side(s) of the tank and pressed into the recess. Note one
pack is supplied as standard. Additional packs can be purchased.
2. Remove the combs with a gentle rocking motion; rinse the well out with ddi H2O to eliminate any
residue.
3. 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.
4. Fill the outer tank with 1 x reservoir buffer. See Appendix for recommended running buffer
solution. Table 6. Shows the volume of buffer required.
Table 6.
Buffer Volume
Vertical Mini
Minimum – Inner tank is filled to above the wells. Outer Tank is
250 mL
filled to just flood the bottom of the glass plates. Cooling potential
is at a minimum which may affect resolution.
Maximum – Inner tank is filled to above the wells. Outer Tank is
1200 mL
filled to the maximum fill line. Cooling is high offering good
resolution of samples.
Using the cooling packs – Inner tank is filled to above the wells.
1000 mL
2 Cooling packs are inserted behind the gels. Outer Tank is filled
to the maximum fill line.
5. Load the samples into the wells using a pipette tip taking care not to damage the wells or induce
any air bubbles.
6. Fill any unused wells with 1X sample buffer.
7. Note the orientation in which the samples are loaded.
Gel Running:
1. Fit the lid and connect to a power supply.
2. Consult Table 7 for details on recommended power supply voltage settings.
3. Run times vary with concentration and protein size. When the dye front is approximately 1 cm,
from bottom, turn off the power supply. If resolving proteins less than 4Kd, the power supply
should be turned off sooner.
10
4. Always unplug the power cord from the power supply prior to removing the lid.
5. Remove the gel running module, first emptying the inner buffer into the main tank.
6. Unscrew the pressure bars to release 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 running buffer
and then gently lifting with a spatula.
7. The gel is now ready for further analysis.
Table 7.
Recommended Voltage and Current settings for 1mm
Voltage.mA
thick, 12% gels.
One gel
90-225V, 20-45mA
Two gels
90-225V, 40-90mA
Three gels
90-225V, 60-135mA
Four gels
90-225V, 80-180mA
The recommended power condition for optimal resolution with minimal thermal band distortion is 150
volts, constant voltage setting. No adjustment of the setting is necessary for thickness or number of
gels. The usual run time is approximately 40-45 minutes. Current should be approximately 50mA per
gel (120mA for two gels) at the beginning of the run. During the 45 minute run, the current will slowly
drop to about 20mA per gel. This drop is caused by the change in buffer ions in the gel, causing a
slow rise in the resistance in the gel. As one would expect from the Ohms law (V=I*R), at constant
voltage (V) a rise in the resistance (R) results in a drop in the current (I).
Protein Blotting using the Vertical Mini Western Blot Insert
Setting up the cassette sandwich: (Common buffer solutions are listed in the Appendix.)
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 fiber pad.
c. Two pieces of .45µm filter paper, pre-soaked in buffer.
d. Gel - cut the left hand corner for indexing,
e. Transfer membrane. Follow manufacturer’s directions for presoaking. Smooth out any air
bubbles that may be trapped under the membrane.
f. Two pieces of filter paper, pre-soaked in buffer.
g. Pre-soaked fiber pad.
h. Cassette clamp +ve (red) side slotted into the groove in the bottom of the black cassette.
Note: do not handle the membrane without gloves.
2. Assemble the fiber pads, filter papers, gel and transfer membrane in the above order and roll with
a pipette to remove any trapped air. Place on the black and red cassette with the membrane
facing the anode (red) side, 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
the Appendix for recommended buffer solutions. Improved transfer can be obtained by using
chilled buffer.
Table 8. shows the volume of buffer required.
Buffer Volume
Vertical Mini
One Cassette
1380 mL
Two Cassettes
1290mL
Three Cassettes
1200mL
Each cooling pack takes the place of 100 mL of buffer.
Blot Running 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 in which the blot
sandwiches were loaded.
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 center 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, attach the lid and connect to a power supply.
12
4. Consult Table 9 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 adjust the
time of the blot to your particular samples and conditions.
5. When the blot time is completed, turn the power supply off and remove the lid of the unit.
6. Remove the cassettes from the main tank.
7. Lift the hinge of each cassette and gently pry apart the blot sandwich and remove the membrane
from the gel.
8. The membrane can now be further processed. Remember to save the filter paper behind the gel
to check for blow through of smaller proteins.
Table 9. Recommended current settings.
Duration of Blot
Vertical Mini
One Hours
40mA
Three Hours
20mA
1St Dimension Electrophoresis using the Vertical Mini 2-D Tube Gel Module
Capillary Tube Gel Pouring:-
13
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 Parafilm.
3. Prepare the following solution. This will be enough to pour twenty 80mm Capillary Tubes. For
Native IEF Gels, do not use Urea or NP40 and use 18 mL of distilled water instead of 16ml;
16 mL Distilled Water (18ml for Native Gels)
2.4 mL Glycerol
0.9 mL 4-8 Resolyte or other available 40% ampholyte solution
3.8 mL Acrylamide/Bis solution
16.2g Urea (omit for Native Gels)
0.6 mL NP40 (omit for Native Gels)
This solution should be degassed prior to pouring.
Add 120 µl of 10% w/v ammonium persulphate solution and 15 µl TEMED and mix well.
4. Using a Hamilton or similar syringe, insert the needle into the tube and carefully inject the solution
so that the tube fills from the bottom. Keep filling to 1cm from the top of the tube. The tubes can
be gently tapped to get rid of air bubbles.
5. Fill the remaining 1cm gap with water saturated isobutanol.
6. Leave to fully polymerize, which will normally take 1 – 2 hours.
7. After polymerization, remove the water-saturated isobutanol. Tube gels can be used immediately
or stored wrapped in a damp paper towel and plasticwrap at 4oC. The Parafilm at the bottom of
the tubes must be removed prior to electrophoresis.
Method 2:- Filling By Capillary Action
1. Place the capillary tubes in a suitable outer receptacle such as a 15 mL flat bottom 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 volumes may need to
be increased.
Prepare the following solution. This will be enough to pour twenty 80mm Capillary Tubes. For Native
IEF Gels, do not use Urea or NP40 and use 18 mL of distilled water instead of 16 mL;
32 mL Distilled Water (18 mL for Native Gels)
4.8 mL Glycerol
1.8 mL 4-8 Resolyte or other 40% ampholyte solution
14
7.6 mL Acrylamide/Bis solution
32.4g Urea (omit for Native Gels)
1.2 mL NP40 (omit for Native Gels)
This solution should be degased prior to pouring.
Add 240 µl of 10% w/v ammonium persulphate solution and 30 µl TEMED, the solution above.
3. Fill the outer tube with 70% of the acrylamide solution. The 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 there is less than a 1cm non-filled space at the
top, remove some of the acrylamide solution from the outer tube until the height is 1 cm from the
top. If there is a greater than 1cm space at the top, add more acrylamide solution. When the
solution has reached to within 1cm of the top of the tube, stop adding the acrylamide solution.
6. Using a syringe, fill the remaining 1cm gap with water saturated isobutanol.
7. Leave to fully polymerize, which will normally take 1 – 2 hours.
8. After polymerization, remove the water-saturated isobutanol. Tube gels can be used immediately
or stored wrapped in a damp paper towel and plastic warp at 4oC.
9. The tubes may have a residual of acrylamide on the outside and may need cleaning with distilled
water before insertion into the tube gel insert.
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 ~ 500 mL of 10mM H3PO4 Anode Buffer 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 Tube Gel Module into the unit and fill the upper buffer reservoir with ~100 mL of 20mM
NaOH Cathode Buffer 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. Dissolve the samples in 1% ampholyte with 20% glycerol.
5. Load the tubes with the samples.
6. Replace the safety lid firmly making sure that the electrical connectors form a good contact.
7. 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
15
8. Run at 400V for 3 hours and then 800V for 30 minutes. 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 Tube Gel 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, or b) inserting a pipette 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 Tris glycine SDS running buffer used for the 2-D phase.
2. Remove the gel(s) from the running buffer pre-soak, and slice the gel in half. Place each
lengthways onto the top of a vertical gel. The gel should be cast using a 2-D comb. See the
section earlier in this manual on vertical gel casting for additional information.
3. Hold the tube gel in place by pouring over it a 1% agarose gel containing the tracker dye.
4. Electrophorese as usual until the tracker dye has advanced the required distance.
5. The samples can be visualized using any of the standard staining methods or alternatively they
can be blotted.
16
Appendix
Stock Solutions for SDS PAGE gels:Stock 30% Acrylamide Gel Solution:30.0 g acrylamide
0.8 g methylene bisacrylamide
Distilled Water to 100 mL
.
Stock 4 X Resolving Gel Tris (1.5 M Tris HCl pH8.8, 0.4 % SDS)
To 110 mL Distilled Water add 36.4 g of Tris base
Add 8 mL of 10 % SDS
Adjust pH to 8.8 with 1N HCl
Adjust the final volume to 200 mL with Distilled Water.
.
Stock 4 X Stacking Tris (0.5 M Tris HCL pH 6.8, 0.4 % SDS)
To 110 mL Distilled Water add 12.12 g of Tris base
Add 8 mL of 10 % SDS
Adjust pH to 6.8 with 1N HCl
Adjust the final volume to 200 mL with Distilled Water
Stock 4 X Tris-glycine tank buffer - SDS
36 g Tris base
172.8 g glycine
Distilled Water to 3 L
1 x Tris-glycine tank buffer - SDS
750 mL of 4 X Tris-glycine tank buffer - SDS
30 mL of 10 % SDS
Distilled Water to 3L
Add Distilled Water to a final volume of 200 mL
10 % APS (ammonium persulphate solution)
0.1 g ammonium persulphate
1 mL Distilled Water
Note ammonium persulfate degrades quickly in solution.
Stock 2 X Sample Buffer
2 mL 50% glycerol
.5 mL 2-mercaptoethanol
4 mL 10% SDS
2.5 mL .5 M Tris -HCL
1 mL 1% Bromophenol blue
ddi H2O to 10 mL
Aliquot into 1.5ml microcentrifuge tubes. Store at -20°C.
17
Membrane Selection
Nitrocellulose
Good binding capacity, proteins bind by hydrophobic interactions
Pore Size
.45µm or 22µm
Western Transfer
Amino acid analysis
Nylon
Microporous membrane modified with strongly basic charged groups
Binds negatively charged macromolecules, DNA or RNA with low background
Pore Size
.45µm
Can Re-probe
Southern Transfer
Northern Transfer
Solid phase immobilization
Enzyme immobilization
Gene probe assays
PVDF
High binding capacity
High hydrophobic binding, solvent resistant
Compatible with protein stains and immunodetection techniques
Pore Size
.45µm or .22µm
Can re-probe
Western Transfer
Protein Sequencing
Amino Acid Analysis
Solid Phase Assay Systems
Buffer Preparation - Proteins
Towbin Buffers
Native Gels
Towbin Buffer pH 8.3
25 mM TRIS, 192 mM glycine, 20% Methanol,
3.0 gm TRIS
14.4 gm glycine
200 ml Methanol
add ddi H2O to 1 liter
Denatured Gels
Towbin Buffer pH 8.3, no Methanol
25 mM TRIS, 192 mM glycine
3.0 gm TRIS
14.4 gm glycine
add ddi H2O to 1 liter
18
Warranty
Labnet International, Inc. warrants that this product will be free from defects in material and
workmanship for a period of one (1) years from date of purchase. If a defect is present, Labnet
International will, at its option, repair, replace, or refund the purchase price of this product at no
charge to you, provided it is returned during the warranty period. This warranty does not apply if the
product has been damaged by accident, abuse, misuse, or misapplication, or from ordinary wear and
tear.
For your protection, items being returned must be insured against possible damage or loss.
IT IS EXPRESSLY AGREED THAT THIS WARRANTY WILL BE IN LIEU OF ALL WARRANTIES OF
FITNESS AND MERCHANTABILITY FOR A PARTICULAR PURPOSE ARE LIMITED IN DURATION
TO 12 MONTHS FROM THE ORGINAL DATE OF PURCHASE.
For research and development use only. Not intended for any animal or human therapeutic or
diagnostic use.
19
Accessories
Additional accessories available. Contact Labnet International for details.
Labnet Cat#
Description
E2010-BM
Blotting Module
E2010-B-FB
Fiber Blotting Pads, Pack of 6 pads
E2010-2D-CT
Capillary Tubes, disposable, Pack of 10.
E2010-2D-BP
Capillary Blanking Ports, Pack of 10
E2010-CP
Mini Cooling Pack
Plates and Spacers
Description
Labnet Cat No.
E2110-NG-2
E2110-PG-2
Notched glass plate, 10 x 10 cm
Glass plate, 10 x 10 cm
E2110-PG-0.75-BS
Glass Plate with bonded 0.75 mm spacers 10 x 10 cm
E2110-PG-1-BS
Glass Plate with bonded 1mm spacers 10 x 10 cm
E2110-PG-1.5-BS
Glass Plate with bonded 1.5 mm spacers 10 x 10 cm
E2110-PG-2-BS
Glass Plate with bonded 2 mm spacers 10 x 10 cm
E2110-NG-0.75-BS
Notched Glass Plate bonded 0.75 mm spacers, 10 x 10 cm
E2110-NG-1-BS
Notched Glass Plate with bonded 1 mm spacers, 10 x 10 cm
E2110-NG-1.5-BS
Notched Glass Plate with bonded 1.5 mm spacers, 10 x 10 cm
E2110-NG-2-BS
E2110-DP
Notched Glass Plate with bonded 2 mm spacers, 10 x 10 cm
Dummy plate, 10 x 10 cm
E2110-0.75-S
Gel spacer, 0.75 mm
E2110-1-S
E2110-1.5-S
E2110-2-S
Gel spacer, 1 mm
Gel spacer, 1.5 mm
Gel spacer, 2 mm
20
Combs
Labnet Cat No.
E2110-12-1
E2110-12-1
E2110-12-1
E2110-10-1.5
E2110-10-0.75
E2110-5-1.5
E2110-5-0.75
E2110-16MC-1
E2110-20-1
E2110-5-1
E2110-20-0.75
E2110-12-2
Well
Thickness Well
width
mm
volume
mm
3.75
1 35ul
3.75
1.5 50ul
Description
12 well comb.
12 well comb
10 well comb
10 well comb
10 well comb
5 well comb
5 well comb
16 well comb
20 well comb
5well comb
20 well comb
E2110-8MC-1
12 well comb
8 well comb
E2110-20-1.5
20 well comb
4
4
4
10
10
2.5
2
10
2
3.75
6
2
21
1
1.5
0.75
1.5
0.75
1
1
1
0.75
2
40ul
60ul
30ul
150ul
75ul
25ul
20ul
100ul
15ul
70µl
1 60µl
1.5 30µl
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