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Sample Preparation & Gel Electrophoresis Troubleshooting

Electric currents, wires, leads, combs, leaks… so many opportunities for trouble. But we still use gels, because electrophoresis remains an effective way to separate proteins — so that the results of antibody-based immunodetection can be fairly unequivocal.

Click on the Sample Preparation & Gel Electrophoresis topics to read about the possible causes and remedies:

No Bands or Gel Front

Possible Cause                                        
Remedy

Proteins have run off the gel
  • Decrease the amount of time the gel is run.
  • Decrease the voltage.
  • Ensure that the leads are in the correct orientation, as the electrophoresis leads to the power supply may be reversed, causing the gel to run upward.
  • Increase the acrylamide percentage of the gel.

Not enough protein was loaded on the gel
  • Load more protein into each well.
  • Ensure that the protein concentration of each sample is accurate.

Sample has diffused away from the well prior to applying power
  • Decrease the time between loading the first well of the gel and beginning electrophoresis. If necessary, run fewer gels and/or fewer lanes at once.

Not enough SDS in the sample
  • Without adequate SDS, the proteins are not negatively charged and will not travel through the cell. Ensure adequate SDS concentration.

Sample Doesn't Sink to the Bottom of the Well

Possible CauseRemedy

Not enough glycerol in sample
  • Increase glycerol concentration.

Sample Leaking Out of Well

Possible Cause                                               
Remedy

Wells damaged during comb removal
  • Remove gel comb carefully to avoid disruption of the wells. Rinse wells gently with electrophoresis buffer prior to sample loading to detect damaged wells.

Wells damaged during sample loading
  • Load sample carefully, ensuring that pipette tip does not touch the bottom or sides of the wells.

Bands are Smeared Vertically

Possible Cause                                               Remedy

Incomplete protein solubility blocks proteins
from entering the gel
  • Ensure adequate sonication/homogenization and centrifugation during cell lysate preparation to remove particulate matter.

Protein degradation
  • Add/increase concentration of protease/phosphatase inhibitors during cell lysate preparation.
  • Avoid repeated freeze/thaw cycles of cell lysate and Western blot samples.

Too much protein loaded

Effect of loading too much protein on SDS-PAGE

Effect of loading too
much protein on
SDS-PAGE
(rightmost lane)
  • Decrease the amount of protein loaded per well. Ensure accurate protein concentration of sample.

Gel run too fast
  • Increase gel run time.

Poor quality or old gel
  • Use a fresh gel.

Too Many Bands

Possible Cause                                               Remedy

Protein degradation
  • Add/increase concentration of protease/phosphatase inhibitors during cell lysate preparation.
  • Avoid repeated freeze/thaw cycles of cell lysate and Western blot samples.

Protein aggregation
  • Ensure adequate DTT concentration, which reduces disulfide bonds.
  • Heat Western blot samples in water bath prior to loading onto gel.

Gel Running Unusually Slowly

Possible Cause                  
Remedy

Buffers too concentrated
  • Dilute buffers.

Current too low
  • Increase voltage of gel apparatus.

Gel Running Unusually Fast

Possible Cause                   Remedy

Buffers too dilute
  • Concentrate buffers.

Current too high
  • Decrease voltage of gel apparatus.

Protein Bands Too Close Together (Not Completely Resolved)

Possible Cause                                              Remedy

Insufficient run time
  • Run gel for longer period of time.

Incorrect gel pore size
  • Use a gradient gel (higher acrylamide percentage on bottom than top) that will adequately resolve a broader size range of proteins.

Leftmost and/or Rightmost Bands of the Gel are Distorted

Possible Cause                                              Remedy

Edge effects
  • Do not leave empty wells. Load wells not being used with a small amount of protein to prevent edge effects on neighboring lanes.

Bands Form Smiling Shapes

Possible CauseRemedy

Excessive heat
Gel run is smiling.

Gel run is “smiling.” This is caused by too high a
voltage. Also, target bands are bleached, caused
by using the detection reagent at too high of a
concentration.
  • Run gel at a lower voltage for longer time. Use chilled buffers and run gel in a cold room or packed on ice.
PCR and Protein Kits
Product NumberProduct NameProduct DescriptionPricing
11483188001First Strand cDNA Synthesis Kit for RT-PCR (AMV)sufficient for 30 reactions (including 5 control reactions), kit of 1 (10 components), suitable for RT-PCR, hotstart: no, dNTPs included
11636090910PCR DIG Probe Synthesis Kitsufficient for 25 reaction (50 μL final reaction volume)
11796828001High Pure PCR Template Preparation Kitpkg of 100 purifications, suitable for DNA extraction
D7440JumpStart™ Taq ReadyMix™ for Quantitative PCRFor probe-based real-time PCR
EHIFI-ROExpand™ High Fidelity PCR Systemsufficient for ≤40 reactions (11732641001), sufficient for ≤200 reactions (11732650001), sufficient for ≤1,000 reactions (11759078001)
FHIFIN-ROFastStart™ High Fidelity PCR System, dNTPack
FHIFI-ROFastStart™ High Fidelity PCR System5 unit/μL, suitable for PCR, High Fidelity PCR, hotstart, Multiplex PCR
FPCR-ROFastStart™ PCR Mastersufficient for ≤100 reactions (04710436001), sufficient for ≤400 reactions (04710444001), sufficient for ≤2,000 reactions (04710452001)
KCQS07KiCqStart® One-Step Probe RT-qPCR ReadyMix™for Bio-Rad, Cepheid, Eppendorf, Illumina, Corbett, and Roche systems
PROTSIL1ProteoSilver™ Silver Stain Kit
R4775REDExtract-N-Amp™ PCR ReadyMix™Ready-to-use 2X PCR Master Mix with Loading Dye
XNATREDExtract-N-Amp™ Tissue PCR Kitsufficient for 10 reactions, sufficient for 100 reactions, sufficient for 1000 reactions, hotstart, dNTPs included
XNAT2Extract-N-Amp™ Tissue PCR Kitsufficient for 100 extractions, sufficient for 100 amplifications
Materials
Product NumberProduct NameProduct DescriptionPricing
AXYPCR0208C100Corning® Axygen® PCR Tube Strips
RPROTKSOL-ROProteinase K, recombinant, PCR GradeSolution from Pichia pastoris
PCRH20-ROWater, PCR Gradeliquid, pkg of 25 mL (03315959001 [1 x 25 ml]), pkg of 25 mL (03315932001 [25 x 1 ml]), pkg of 100 mL (03315843001 [4 x 25 ml])
MP0025Venor™ GeM Mycoplasma Detection Kit, PCR-based
DNTPCA1CleanAmp™ dNTPHot-start dNTP mix for improved PCR
1.15444Coomassie Brilliant blue G 250 (C.I. 42655)for electrophoresis
E3004Extract-N-Amp™ PCR ReadyMix™Amplifications to support Extract-N-Amp Plant and Extract-N-Amp Tissue
1.04169Glycinebuffer substance for electrophoresis
MP41G15mPAGE® 4-12% Bis-Tris Precast GelL × W × wells 10 cm × 8 cm × 15 well
A4929N,N′-Bis(acryloyl)cystamineBioReagent, suitable for electrophoresis
RSBREADYBLUE® PROTEIN GEL STAIN
R4526Reference Dye for Quantitative PCR100 ×, solution
1.07673Silane A 174adhesion promoter
S5692SYPRO® Orange Protein Gel Stain
S4942SYPRO® Ruby Protein Gel Stain
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