Make your own free website on Tripod.com
PROTEIN GEL ELECTROPHORESIS TIPS AND TROUBLESHOOTING GUIDE

TIPS FOR SUCCESSFUL GEL ELECTROPHORESIS

1. Use high quality acrylamide and bis.

2. Typical Laemmli sodium dodecyl sulphate (SDS-PAGE) systems includes SDS in both the gel and running buffer (1). However, it was found that molecular weight determinations could be accurately performed with gels lacking SDS if both the running buffer and sample buffer contain SDS. Since excess SDS forms micelles which can interfere with accurate Rf determinations it may be advantageous to limit the amount of SDS in the gel. Also, single % gels lacking SDS could be stored for longer periods than normal gels. If the gel is enclosed in a sealed bag containing tissue paper soaked in 0.02% sodium azide it could last for up to 1 month.

When working with large sample volumes (30 microliter) the SDS concentration should be limited to prevent excess micelle formation which results in diminish resolution. SDS concentration should not exceed 200 microgram/30 microliter sample. Also it has been found that high concentrations of SDS may interfere with Coomassie blue staining times. SDS grade is of utmost importance. We have found that a protein stained background along individual gel tracts with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS. When fresh high quality SDS was used with the same sample sharp protein bands were observed.

3. The stacking gel length should be 1 cm from the well bottom to the top of the separating gel for proper stacking of the protein sample.

4. Band resolution could be improved by doubling the salt concentration in stacking and separating gels, but the gel must be run at lower voltages.

5. To avoid edge effects, add 1x sample buffer to unused wells.

6. If electrophoresis is carried out at low temperatures use lithium dodecyl sulfate (LiDS) instead of SDS. LiDS does not precipitate at low temperatures.

7. During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution.

8. Add 0.1mM thioglycolic acid to upper gel buffer if proteins will be subjected to sequence analysis.

9. When doing protein renaturation or sequencing applications, leave gel for at least 5 hours post-polymerization to allow the ammonium persulfate and the TEMED to react with the gel components which reduces their chance of reacting with the amino-terminal end of the peptide.

10. If the sample is too dilute add trichloroacetic acid (TCA) to 10% (w/v) and incubate for 5 minutes at 4C. Centrifuge and wash pellet with cold acetone. Resuspend pellet in volume desired.

11. If salt concentration of sample is high concentrate protein as in number 10 and resuspend in appropriate buffer. High salt concentrations causes gel artifacts.

12. Although constant power conditions is still used by some scientists for electrophoresis, Hames (2) found that only constant voltage gives constant protein mobility during electrophoresis.

13. Centrifuge all samples in a microfuge tube at 12,000 g for 2-5 mins prior to loading (to remove any aggregates).

14. Never overfill wells. This could lead to artifacts.

15. Always check the protein concentration before loading. Mini-gel wells should not contain more than 150 microgram of protein even for complex mixtures of proteins.

16. Prepared samples (i.e. those which were boiled in sample buffer) could be aliquoted and stored at -20C for 3-4 weeks or at 4C for at least a week. Repeated freeze-thawing results in protein degradation. Before using these samples they should be warmed at 37C for a few minutes to redissolve SDS which precipitates out of solution.

17. For the best resolution of proteins, two-dimensional gel electrophoresis (3) should be utilized.

18. A proper record should be kept for every gel and possibly a gel electrophoresis record made up similar to the one shown in the Appendix. Assign each gel a gel number.

*****

PRECAUTIONS NEEDED

1. Wear gloves at all times when using acrylamide. Acrylamide is a neurotoxin. Even after polymerization some unpolymerized acrylamide may be present. If the skin should come into contact with acrylamide solution or powder, wash immediately with soap and copious water.

2. The high electrical power used in gel electrophoresis is very dangerous as such one should never disconnect the electrodes before first turning off the power supply.


TROUBLESHOOTING GUIDE

1. Poor resolution

a) Sample volume too large. Concentrate samples.

b) Excess micelle formation. Do not exceed 200 microgram SDS/30 microliter sample.

c) Refer to numbers 3,8-11 and 13.

2. Run taking unusually long time

a) Buffers too concentrated. Check buffer protocol; dilute buffer if necessary.

b) Current too low. Increase voltage by 25-50%.

3. Run too fast, poor resolution

a) Buffers too dilute. Check buffer protocol; concentrate buffer if necessary.

b) Current too high. Decrease voltage by 25-50%.

4. More bands than expected observed for a purified protein

a) Proteolysis. Minimize the time between sample preparation and electrophoresis.

5. Fewer bands than expected with a heavy band at the dye front

a) Gel percentage is too low for the molecular weight range of the protein sample. Use a higher percentage acrylamide gel (increase % T in resolving gel).

6. Doublets observed where a single protein band is expected on SDS-PAGE

a) A portion of the protein sample may have re-oxidised during the run, or may not have been fully reduced prior to run. Prepare fresh sample solution using fresh -mercaptoethanol or dithiothrietol (DTT). Increase -mercaptoethanol or DTT concentration in the sample buffer.

7. Artifact band observed at approx. 67kDa in reduced samples, especially with silver staining

a) Excess reducing agent (-mercaptoethanol). The addition of iodoacetamide to the equilibration buffer just before applying the sample to the gel has been shown to eliminate these artifact bands.

b) Skin protein contaminants. Use new electrophoretic solutions and wear gloves when handling and loading the gel. More common when highly sensitive stains are used.

8. Skewed or distorted bands

a) Poor polymerization around sample wells. Increase ammonium persulfate and TEMED concentrations by 25%.

b) High salt concentration in sample. Remove by dialysis, Sephadex G-25 or any other desalting column or by Amicon concentrators.

c) Excessive pressure applied to the gel plates when the gel is placed into the clamp assembly. Do not overtighten the screws on the clamp assembly.

d) Uneven gel interface. Use a spirit level to make sure the gel apparatus is even. Overlay separating gel with water carefully.

e) Uneven heating of the gel. Either use a cooled apparatus or reduce the current at which electrophoresis is performed.

f) Insoluble material in the gel or inconsistent pore size throughout gel. Filter gel reagents before use and ensure that the gel mixture is well mixed and degassed before pouring the gel.

9. Lateral band spreading

a) Diffusion of sample out of the wells before the power was turned on. Minimize the time between sample application and power start-up.

b) Diffusion during migration through the stacking gel. Increase voltage by 25% during stacking gel or increase %T of stacking gel by 1%.

10. Vertical streaking of protein

a) Sample precipitation. Centrifuge all samples before loading wells. If problem still persists decrease %T of separating gel.

b) Sample overload. Dilute sample or reduce voltage by about 25% to minimize streaking.

11. Protein band curves upward at both sides of the gel. "Smile effect"

a) Center of the gel running hotter than either ends. Decrease power setting. Check buffer protocol to ensure it is properly formulated.

12. Same protein observed in several neighboring lanes

a) Samples from one well has contaminated adjacent wells. Use a Hamilton syringe to load wells and reduce the sample volume.

b) Do not delay while loading wells. A full well left next to an empty well would eventually contaminate the empty well over time.

13. Diffuse tracking dye

a) Decomposition of sample solution and/or buffer stock solution. Prepare fresh reagents.

14. Diffuse protein bands

a) Diffusion due to slow migration. Increase voltage by 25-50%. Check buffer to ensure it was properly prepared.

b) SDS or sample buffer too old. Prepare fresh solutions.

c) Protein sample not equilibrated. Equilibrate sample to running conditions.

d) Poor quality acrylamide or by resulting in incomplete catalysis. Use electrophoresis grade reagents.

e) Problems in sample preparation. Check to make sure sample is heated to at least 90C for 2 min before loading.

15. Inconsistent relative mobilities

a) Incomplete catalysis. Excessive TEMED or ammonium persulfate. TEMED and ammonium persulfate should be 0.05%.

b) The constituents of the gel may vary in quality from batch to batch or with age. Use one batch of a chemical for as long as possible. Replace aged stock solutions and reagents.

c) The protein amounts loaded differ greatly. Do not overload the gel. Keep the loadings roughly similar in size.

16. Aggregation of proteins

a) Some samples aggregate on boiling. Treat sample at lower temperature (60C).

b) Formation of disulfide bonds between protein in a complex mixture because of insufficient reducing agent. Prepare new sample buffer.

17. Band Streaking

a) High salt concentration. Precipitate and resuspend in lower salt buffer.

b) Sample too concentrated or not enough SDS. Dilute the sample with more SDS solution.

18. Heavily Stained band at gel origin

a) Gel concentration too high. Use lower gel concentration.

b) Aggregation of protein sample prior to electrophoresis. Refer to no. 16.

19. Aberrant Molecular Weight determination High molecular weight

a) Proteins with >10% carbohydrate bind less SDS. Check for carbohydrate.

b) Hydrophilic proteins bind less SDS.

c) Potassium or divalent cations present in sample precipitate SDS. Precipitate sample and resuspend in different buffer.

20. Aberrant Molecular Weight determination Lower Molecular Weight

a) Hydrophobic proteins bind more SDS.

b) Incomplete disulfide bond dissociation because of insufficient reducing agent. Prepare new sample buffer and samples.

21. Gelling time too long

a) Too little ammonium persulfate or TEMED. Increase both by 50%.

b) Temperature too low. Cast at room temperature.

c) Old ammonium persulfate and TEMED. Use fresh ammonium persulfate and new TEMED.

d) Poor quality acrylamide or bis. Use electrophoresis grade acrylamide and bis.

e) High concentration of thiol reagents. High concentrations of thiol reagent inhibit polymerization. Use less thiol reagent.

f) Omission of a reagent from the gel mixture. Have a list of all the reagents required and thick off reagent when utilized.

g) Incorrect concentrations of prepared reagents. Check protocol. Make up new reagents.

h) Degassing the acrylamide solution leads to a more rapid polymerization. However I have found that this step is cumbersome and unnecessary for most applications.

22. Gel too soft

a) Poor quality acrylamide or bis. Use electrophoresis grade acrylamide and bis.

b) Too little crosslinker. Make sure proper %C.

23. Gel does not polymerize

a) Temperature too low. Cast at room temperature.

b) Too little ammonium persulfate or TEMED. Increase both by 50%.

c) Poor quality acrylamide or bis. Use electrophoresis grade acrylamide and bis.

d) Ammonium persulfate or TEMED are old. Use fresh ammonium persulfate and new TEMED.

24. Swirls in gel

a) Excessive catalysis. Gel polymerizes in less than 15 minutes. Reduce ammonium persulfate and TEMED by 25% each.

b) Gel inhibition. Polymerization time >1 hour. Increase ammonium persulfate and TEMED by 50%.

25. Gel brittle

a) Too much crosslinker. Check protocol for %C. Recheck solution and weights.

26. Gel turns white

a) Bis concentration too high. Recheck solution or weights used.

27. Upper buffer chamber leaks

a) Upper buffer chamber over filled.

b) Improper assembly. Check assembly.

28. Leaking during gel casting

a) Chipped glass plates. Check glass plates for flaws. If minor flaws at bottom of glass plate parafilm could be used to properly seal the glass plates.

b) Improper alignment of gel plates. Check to ensure that the spacers and plate bottoms are flush.

29. Gel cracking during polymerization

a) Excess heat generation. Use cooled reagents.

30. Samples do not sink to bottom of well

a) Insufficient glycerol in the sample buffer. Recheck protocol.

b) Combs removed before stacking gel properly polymerized. Let stacking gel polymerized for 30 minutes before removing combs.

31. Sample preparation yellow in colour

a) Solution acidic: add NaOH until the solution turn blue.

b) Too little bromophenol blue in sample buffer.

32. Detachment of slab gels from glass plates during gel electrophoresis.

a) Inadequately cleaned glass plates

33. Base of sample well appears to be dragged downwards in the direction of electrophoresis.

a) Could be due to trapping of high molecular mass, high charged species at the gel surface. Very common when high concentrations of nucleic acid is present in the sample.

b) Check sample for nucleic acid and remove if present in significant quantities in the sample.

34. Poor Sample Wells

a) Distorted or broken wells are formed when the comb is not removed carefully. Comb should be removed only in a vertical manner.

b) When stacking gel resists the removal of the comb use a gel of lower % T.

c) When the wells contain a loose webbing of polyacrylamide it is likely that the comb fits loosely or the gelling rate is too fast. Replace the comb with a tighter fitting one and check the amount of TEMED and ammonium persulfate being added.

35. Gel Cracking during electrophoresis

a) The running conditions are too warm. This is especially common with high percentage gels.

36. Detachment of the gel from the glass plates

a) Probably due to unclean plates. After being rinsed with distilled water, they must drain cleanly without water spots.

37. Bands on part of the slab do not move down the gel

a) This is usually due to air bubbles between the plates underneath the affected lanes. Make sure no bubbles are present in the gel when pouring.

38. Formation of a sticky top on the gel

a) Penetration of the gel by butan-2-ol. Overlay the gel with butan-2-ol without mixing them. Do not leave butanol-2-ol to stand overnight on a polymerized gel or use water instead of butan-2-ol.

39. Protein bands are not sufficiently resolved

a) Insufficient electrophoresis. Prolong the run.

b) The separating gel's pore size is incorrect for the proteins that need to be separated. Alter the %T and/or %C of the separating gel appropriately.

40. Protein bands are not of uniform thickness

a) The sample was loaded unevenly. Check that the sample well bottoms are straight and horizontal.

STAINING AND DESTAINING

41. Non-specific Coomassie blue staining

a) Decomposition of undissolved dye. Filter dye solution.

42. Protein bands not seen properly

a) Coomassie stain not sensitive enough. Gel can be rinsed and subsequently silver stained.

b) Not enough protein loaded onto the gel. For Coomassie blue stained gels each protein band needs at least 0.5 microgram of protein to be sufficiently stained.

c) Volume of Coomassie Blue too little. Increase the volume of staining solution to dilute out the SDS present in the gel.

d) Use a more concentrated staining solution and longer staining time.

e) Check the concentration of methanol (which strips SDS from the protein) used in the staining solution. Increase the methanol concentration if necessary.

43. Uneven staining of gels

a) Incomplete penetration of the dye. Leave gel in stain for a longer time.

b) Not enough dye.

c) Agitation was insufficient. Agitate when staining.

d) High concentration of SDS may interfere with coomassie blue staining.

44. Metallic sheen on gels after staining with Coomassie Blue

a) Solvent was allowed to evaporate causing the dye to dry on the gel.

45. A thin layer of Coomassie Blue on gel surface after destaining

a) Can easily be removed by a quick rinse in 50% methanol or by gently swabbing the gel surface with destain-soaked tissue paper.

46. Blotches near gel borders and over gel

a) Could be due to gel handling without gloves

47. Continuous stained region from the gel origin to near the buffer front.

a) Contamination of sample buffer. Make fresh sample buffer.

48. High silver staining background

a) Acrylic acid contamination in the acrylamide and/or bis-acrylamide. The highest quality reagents should be used.

b) Backgroung staining that is associated with silver detection of proteins in polyacrylamide gels has been shown to be due mostly to the amide groups in the crosslinker bisacrylamide (4). If the background staining is a serious problem use diacrylylpiperazine as the crosslinker. This crosslinker provides improved electrophoretic separation of proteins together with reduced background.

49. Stained bands become decolourized

a) Over-destained gel. Restain the gel and reduce the destaining time.

DRYING GELS

50. Cracking of gels during drying under vacuum

a) Vacuum released before gel is properly dry.

b) Slab gels > 1.5mm are being used.

c) Gel was allowed to swell before drying.


Electrophoresis Links

American Electrophoresis Society (known as The Electrophoresis Society) :
British Electrophoresis Society (BES) :
Swiss Electrophoresis Society:
Japanese Electrophoresis Society (JES):
French Electrophoresis Society :
SWISS-2DPAGE Protein Database :
My Homepage:
Aldrin's Biochemistry Resources:

Aldrin V. Gomes, Biochemistry Unit, Faculty of Medical Sciences, The University of the West Indies, St. Augustine, Trinidad and Tobago


This page has been visited times.