SE 260 Mini-Vertical Unit for Two Slab Gels, complete

The gel pore size is too small

Decrease the % T (Total acrylamide concentration) of the resolving gel

The protein has precipitated or aggregated

Heat the sample at a lower temperature (70 °C or less) for 1–2 minutes

Sample contains particulates

Centrifuge or filter the sample before loading to remove particulates

Sample contains salt

Dialyze or desalt the sample

Too much sample

Load less sample

Proteolytic degradation of sample

Add protease inhibitors such as PMSF (Phenylmethyl-sulfonyl fluoride) if necessary to prevent proteolytic degradation of sample

Sample preparation has to be checked

Check sample preparation:

1. Dialyze or desalt the sample.

2. Centrifuge or filter the sample to remove particulates.

Gel preparation and polymerization have to be checked

Check gel preparation and polymerization:

1. De-aerate the stacking gel solution and avoid trapping air bubbles under the comb teeth.

2. Overlay the resolving gel monomer solution with water-saturated n-butanol to avoid forming an uneven gel surface.

3. After polymerization is complete and the comb is removed, wash out the well with running buffer to remove polyacrylamide particulates and also to stop continued polymerization of the acrylamide solution at the bottom of the well.

Oxygen is present

1. Degas the stacking gel solution and avoid trapping air bubbles under the comb teeth.
2. Overlay the running gel with water-saturated n-butanol before polymerization begins to avoid forming an uneven gel surface.

Sample contains particulates

Centrifuge or filter the sample before loading to remove particulates

Sample contains salt

Dialyze or desalt the sample

1. Avoid stressing the ears by only handling the sandwiches by the sides and bottom.

2. Separate sandwiches along the bottom of the stack.

Glass plates have been exposed to solutions with too high alkali concentration

1. Avoid extended exposure of glass to alkali (high pH) solutions

2. Do not leave plates standing in a Decon 90 solution for longer than overnight as this will eventually cause etching due to the alkali nature of Decon.

Clamp screws tightened too much, stressing the glass

The screws need not be tightened more than finger-tight to ensure a seal

Plates are stressed by excessive heat generated from the electrophoresis

Run gels slower

Foreign material lies on the spacers

Check that no foreign material lies on the spacers before applying the clamps

UBC is warped or shrunken which would happen if the unit had been run at temperatures exceeding 50 °C without proper cooling, or if the unit had been autoclaved

Cooling requires an external cooling water recirculator, such as the MultiTemp III.

Preventive action: "Proper cooling" does not mean running tap water through the UBC

Foam gasket doesn't seal correctly

1. Check that the diameter of the foam gasket is 4.5 mm. Replace, if necessary

2. Check that the foam gasket is still supple and free of micro-cracks. If not, remove and replace the gasket.

Preventive action: Do not stretch the foam gasket when inserting the gasket into its groove as this reduces the diameter of the gasket.

The instrument has been exposed to anti-freeze or organic solvent

Circulate only water or 50/50 water/ethylene glycol through the heat exchanger

Unregulated pressure of coolant source

Only use coolant sources with regulated constant pressure

Too high buffer temperature, temperature above 45ºC

All plastic parts are designed for a maximum temperature of 45ºC

1. Circulate coolant through the heat exchanger during electrophoresis to minimize heating.

2. Perform passive cooling actions by chilling the buffer before use, running the unit in a cold room, or both

1. Check power supply. Try another power supply to verify that the problem does not lie with the power supply.

2. Check current continuity from lid to plugs of both the black and red leads with a multimeter or current meter (ammeter).

3. Check that the lid sockets are firmly connected to the 2 electrode posts.

4. Check current continuity from the electrode posts to the ends of the platinum wires with a multimeter or current meter (ammeter).

5. Check glass plate sandwich setup. The notched plate should be facing towards the cooling core, then current can go from platinum wire via UBC buffer to the top of the gel.

6. Ensure that UBC buffer level is high enough to contact the top of the gel.

7. Ensure that LBC buffer level is high enough to contact both the bottom of the gel and the bottom of the cooling core.

Gels are not identical concerning % acrylamide, % crosslinking, buffer content or thickness

Current splits evenly between the 2 gels only if the conductivity of the 2 gels are perfectly identical.

1. The gels must be identical in % acrylamide, % crosslinking and buffer content.

2. The gels must have identical cross-sectional area. In other words, the gels must be of the same thicknesses.

Running buffers in the two upper chambers are not identical

The running buffers in the two upper chambers must be identical

Also make sure buffer in the upper chamber is high enough to contact the top of the gels

Samples contribute differently

Samples can contribute conductivity. High salt levels (or buffer concentrations) can increase the conductivity of one gel relative to the other.

Solutions need to be adjusted

Adjust the solutions:

1. Check recipes, gel concentrations, and solutions. (For instance, do not use Tris-HCI instead of Tris base.)

2. If proteins move too fast dilute the buffer so that less current will be carried.

3. If proteins move too slow, increase the buffer concentration so that more current will be carried.

4. If the required pH of a solution is exceeded, do not back-titrate. Prepare fresh buffer.

5. Dispose of older acrylamide solutions and use only stock of the highest quality.

6. Only use freshly deionized urea.

Too high salt concentration in samples

Decrease the salt concentration of samples

Particles in UBC

Check the UBC. It should be free of particles.

Electrical leakage

1. Check the alignment of the glass plates and spacers. Make sure there is no dirt or grease on the plates or spacers, and no cracks in the glass.

2. Check the heat-exchanger for leakage around the grommets.

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Adjust the voltage or current settings: To increase or decrease the migration rate, adjust the voltage or current by 25 - 50%.

Sample probably contains some nonionic (or cationic) detergent that is competing with the SDS (Sodium dodecyl sulfate) to coat the proteins

It is the negative charge of SDS that causes the proteins to migrate to the anode. Samples with Triton X-100 will lower the charge density and cause the sample proteins to migrate slower. The markers, coming out of a separate vial, probably don't contain Triton and will therefore migrate correctly. The solution is to clean the sample to remove the nonionic detergent (try TCA-acetone precipitation or the SDS-PAGE Clean-Up Kit). TCA=Trichloroacetic acid

Incorrectly prepared buffer, either in the buffer chambers or in the gel

Make up fresh buffers and re-check gel casting recipe.

Electrical leakage

Check the alignment of the glass plates. Make sure there is no dirt or grease on the plates or spacers, and no cracks in the glass.

Too much bisacrylamide.

Crosslinker should be at 2.6 %C for standard SDS (Sodium dodecyl sulfate) gels where:

%C = (g bis x 100)/(g monomer + g bis)

Poor quality of reagents

Use only the highest quality reagents

Issues related to Urea

Only use freshly deionized urea.

Low molecular weight species have diffused

Begin electrophoresis as soon as the sample is loaded to prevent low molecular weight species from diffusing.

Poor stacking

1. Only use gels that were recently prepared

2. Add a stacking gel or increase height of the stacking gel. Prepare the resolving-gel surface by first rinsing it with stacking gel monomer before pouring the stacking gel to ensure continuity between the gels.

3. Check pH values of the separating and stacking gel solutions. Do not backtitrate buffers.

Incomplete gel polymerization

Allow the gel to polymerize fully

Running conditions

Conduct the separation at a lower current or voltage setting

Too much TEMED or APS (Ammonium persulphate)

Lower the amount of TEMED or APS

Sample incorrect prepared

Sample preparation:

a. Dialyze or desalt the sample.

b. Centrifuge or filter sample before loading to remove particulates

c. Reduce the sample volume or concentration.

d. Improve dissociation of protein subunits by heating sample in SDS (Sodium dodecyl sulfate) sample buffer 1-2 minutes at 100 °C. Store on ice after heating.

e. Store sample on ice before it is denatured.

f. Add protease inhibitors such as PMSF (Phenylmethyl-sulfonyl fluoride) if necessary to prevent proteolytic degradation of sample.

g. Add more mercaptoethanol or dithiothreitol; check sample treatment.

h. Make fresh SDS sample buffer

i. Use the same buffer for the sample as for the stacking gel.

j. Increase the amount of glycerol or sucrose to increase sample density.

k.Store samples to be frozen in aliquots to prevent repeated freezing and thawing.
Store at -40 °C to -80 °C.

l. In a continuous buffer system, the sample may be too dilute. Use a discontinuous buffer system with a stacking gel, or a more concentrated sample.

Too high running temperature

Reduce the running temperature:

1. Pre-chill the buffer.

2. Decrease the current or voltage setting. (For Laemmli gels: 10 mA per 0.75 mm thick gel, 15 mA per 1.5 mm thick gel.)

3. Conduct electrophoresis in a cold room.

4. Fill the tank to the maximum (marked) level

Water is impure

Use only double-distilled water

Dirty plates e.g. fingerprints

Soak plates in a strong laboratory detergent and rinse well in distilled water. Please use gloves.

Plates are scratched

Replace glass plates. To some extent the effect of scratches can be counteracted by treating plates with silanizing reagent such as Repel-Silane.

Plates were stored with, or soaked together with, plates that were pre-treated with Bind-Silane.

Always segregate Bind-Silanized plates. Bind-Silanized plates can "contaminate" untreated plates if placed in contact, or soaked together. Glass plates can be re-used after scraping off the polyacrylamide gel and thoroughly washing the glass plates with strong sodium hydroxide solution.

Too much catalyst: gel polymerized in < 10 min

Reduce both APS (Ammonium persulphate) and TEMED by 25%.

Not enough catalyst: gel polymerized in > 50 min

Increase both APS (Ammonium persulphate) and TEMED by 50%

Solutions not mixed

Mix thoroughly after adding TEMED

Gel concentration

Near the buffer front. Molecules are not sufficiently restricted by the resolving gel pore size: increase the %T (Total acrylamide concentration).

Near the top of the gel when the buffer front has reached the bottom. The gel pore size is too small: decrease the %T of the resolving (or stacking) gel.

At both top and bottom of the gel. The molecular weight range of the sample requires an acrylamide concentration gradient to resolve the full range of protein sizes.

Protein has degraded

Use protease inhibitors during the isolation step to prevent protein degradation by proteases.

Protein has precipitated

The protein has precipitated. Heat the sample at a lower temperature (70 °C or less) for 1–2 min.

Poor chemicals

1. Use only recent stock of the highest quality reagents.

2. If the dry ammonium persulphate does not crackle when added to water, replace with fresh stock.

3. Increase TEMED or APS (Ammonium persulphate) concentration, or both.

Solutions with extreme pH values (especially acidic) may not polymerize.

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Oxygen is present

Remove oxygen from the gel environment: Degas the monomer solution 5 to 10 minutes before pouring and then overlay the gel surface with water-saturated n-butanol.

Gel solution has too low temperature

Adjust the gel solution temperature to a minimum of 20 °C, especially for low %T (Total acrylamide concentration) gels.

Too low concentration of TEMED or APS (Ammonium persulphate)

Increase TEMED or APS concentration, or both

Not enough crosslinker

Crosslinker should be at 2.6 %C for standard SDS (Sodium dodecyl sulfate) gels where:

%C = (g bis x 100)/(g monomer + g bis)

Too much bisacrylamide

Check concentrations of solutions

The pH of the stacking gel or running buffer is incorrect

Check buffer stocks and casting recipe.

Too high sodium or potassium concentration.

Avoid using solutions with a high sodium or potassium concentration

Issues with the acrylamide solution

1. Check the expire date of the acrylamide solution
2. Use only the highest grade of acrylamide

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For best results, allow a stacking gel height of 2.5 times the height of the sample in the well