miniVE, complete

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

Gel pore size is too small

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

Protein precipitates or DNA becomes denatured

Decrease the temperature at which the sample is prepared to 70 °C or less, and limit exposure to heat to 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

Protein or nucleic acid is not sufficiently restricted by the resolving gel

Increase the %T (Total acrylamide concentration)

Air bubbles in the transfer stack

Remove all trapped air bubbles in the transfer stack

Preventive action: Take care to not introduce air bubbles between the layers of the stack during stack assembly.

Stack is too loose

Make sure that the stack is not loose. A loose stack will result in smeared bands, or blank areas. Add more sponges to make the stack sufficiently tight.

Dacron sponges are getting compressed

1. Over time, the Dacron sponge will get compressed and needs to be replaced

2. Use the foam sponges to TE22. The foamed sponges and Dacron sponges have the same dimensions.

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Use a lower ionic strength buffer

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Check electrode continuity

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Increase the transfer period. (Try doubling it. CAUTION: the Towbin buffer contained within a blot module contains sufficient ionic strength for transfer up to 2 hours. Do not exceed this length of time or the buffer will become depleted

Over heated buffer

Make sure the temperature of the buffer in the blot module does not become overheated. As temperature of the transfer buffer increases, so does conductivity. Higher conductivity means ever higher temperature and you are soon in a positive feedback loop where the buffer is brought to a very high temperature. Should the temperature be high enough, you will simply be "cooking" your proteins in your gel--denaturing them and making them difficult to migrate out. Use passive cooling (fill the tank with ice water) and monitor the temperature periodically during transfer.

Gel has been exposed to staining or fixing agents before transfer

Do not expose the gel to staining or fixing agents before transfer.

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Try one or several of the following suggestions:
Use a thinner gel
Use reagent-grade chemicals
Reduce the gel acrylamide concentration
For proteins, use 3.5 mM SDS (0.1%) in the transfer buffer
Increase the net charge on the protein by changing to a transfer buffer with a different pH. Lower pH (<6 - 7) increases the positive charge on proteins; higher pH (>6 - 7) increases the negative charge on proteins.

Presence of methanol

Methanol shrinks polyacrylamide gels, making it more difficult for proteins to exit. Avoid including methanol in the protein transfer buffer, or reduce the amount to the absolute minimum.

If this occurs from the start, then the connections to the power supply are reversed

Change the connections to the power supply

If this occurs after some time into a long run, then the tank buffer is depleted

If tank buffer is exposed to electrical current for a long time and the tank buffer is not recirculated, then the pH of the buffer changes near the electrodes (because protons are being reduced to hydrogen gas at one electrode, and hydroxide ions are being oxidized to water and oxygen at the other). When this happens, the dye will become positively charged relative to the more negative environment at the anode, and start migrating in the "wrong" direction (i.e., to the cathode).

After a few hours of a run, turn off the power supply, and then mix the buffer at the cathode side with the buffer at the anode side (or replace both reservoirs with fresh buffer). Reconnect the power supply and continue the run.

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

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)

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.

Incomplete polymerization

Ensure that polymerization is complete

pH of buffers is off which is why stacking is not occurring.

Prepare fresh SDS (Sodium dodecyl sulfate) running buffer and gel buffers

Sample has degraded

To avoid or retard sample degradation, store on ice and fully denature the sample

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Try the following:

1. Use only high quality acrylamide and bisacrylamide

2. Make fresh sample buffer.

3. Add a protease inhibitor such as PMSF (Phenylmethyl-sulfonyl fluoride)

Stacking is not occurring due to incorrect buffer pH

Prepare fresh SDS (Sodium dodecyl sulfate) running buffer and gel buffers

Sample doesn't contain same buffer as stacking gel.

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

Too much TEMED or APS (Ammonium persulphate)

Reduce TEMED or APS concentration by 25%

Discontinuity between both gels

Prepare the separating gel surface by rinsing it with stacking gel monomer before pouring the stacking gel to ensure continuity between both gels

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

Field strength too high

Decrease the field strength

Transfer period too long

Decrease the transfer period

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Include methanol in the protein transfer buffer (up to 20% for nitrocellulose, or up to 10% for PVDF). PVDF=Polyvinylidene difluoride

Please note that methanol shrinks polyacrylamide gels, making it more difficult for proteins to exit.

Water in the outside tank too warm or no water in tank

1. Fill the outside tank with cold water at the start of the transfer? There is no active cooling with this unit, and thus temperature should always be monitored.

2. At intermediate points during the transfer exchange the now-warm water in the outside tank with fresh ice water.

Set current is too high

Since the miniVE blot module's cross-sectional area is small (roughly 85 cm²) you would use less current to get the same amount of transfer as larger instruments e.g. TE42. 150 mA (or less) for 2 hr should be sufficient. For instance to blot at 400 mA for 2 hr using a miniVE would result in smaller and medium-sized proteins blowing through the membrane.

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