SE 600 Ruby Standard Dual Cooled Vertical Unit, complete

Dirty or damaged components

1. Plates, spacers, and the gasket must be completely clean. Wash if necessary.

2. Replace chipped plates (especially if chipped near the spacers).

3. Check the caster gasket for cuts or cracks and replace if necessary.

Mis-aligned parts

Check plate and spacer alignment and realign if necessary

Over-clamping

1. Turn cam only as far as necessary to create a seal (usually 90–150°, but up to 180°).

2. On each spacer apply a light film of GelSeal compound to the bottom outside corner only. Do not use silicone grease.

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

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

Air bubbles

Remove air bubbles before inserting combs. Slide comb into solution at an angle. If comb must be removed, add more monomer solution before reinserting the comb.

Incomplete or delayed polymerization

Allow acrylamide gels to set for a minimum of 1 h.

Debris in wells

Rinse out unpolymerized gel with sample buffer

Comb removal

1. Remove the comb at a slight angle and very slowly to prevent damaging the gel.

2. Agarose gels: Lower the comb no more than 1 cm into the gel.

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

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

Electrical leakage

1. 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.

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

Electrical path to outside ground/earth

1. Add more silicone grease to seal heat exchanger grommets.

2. Check for leaks or cracks in the heat exchanger. Replace worn grommets.

Over-clamping

Turn the cams only as far as necessary to create a seal (usually 90 to 150°, but up to 180°).

Worn caster gasket

Check the caster gasket for cuts or cracks and replace if necessary. Also, make sure the gasket is correctly seated in place.

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Apply a light film of Gel Seal to the bottom outside corner of each spacer. Do not use silicone grease.

Mis-aligned parts

Check plate and spacer alignment and realign if necessary

Chipped glass plates

Check if plate edges (bottom) are chipped and replace chipped glass plates as needed

Plates, spacers, and gasket not clean enough

Plates, spacers, and the gasket must be completely clean. Wash if necessary. (Wash spacers and gaskets with mild detergent; glass plates with cleaning solution. Rinse well with tap, then distilled, water.)

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)

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

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

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 gasket is not damaged or pinched, and is still supple and free of micro-cracks. If not, remove and replace.

2. Check that gasket is centered and fits along the upper chamber groove

Mis-aligned parts

1. Check that the glass plates, spacers, and clamps are aligned and fit snugly into the upper chamber gasket.

2. Check that both gaskets are centered and that the positioning ridges fit inside the grooves

Dirty or damaged components

Check that the gasket is not damaged or pinched. Replace if necessary. Check that the upper buffer chamber is not warped from prior exposure to excessive heat.

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

2. Separate sandwiches along the bottom of the stack.

Current leakage around the gel

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. If used, the buffer dam must be secure

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

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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%.

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

Uneven heat distribution

1. Fill the lower buffer chamber to the level appropriate for the run.

2. Use magnetic stirrer and stir bar to keep buffer well mixed.

Excessive heat

1. Circulate external coolant.
2. Prechill the buffer.
3. Decrease the current or voltage setting.
4. Run the gel in the cold room.