Core Bioreagents

DNA / Protein Electrophoresis and Troubleshooting Tables

Effective Range of Separation of DNA in Polyacrylamide Gels

% Acrylamide (w/v)1 Efficient Range of Separation (bp)
3.5 1000-2000
5.0 80-500
8.0 60-400
12.0 40-200
15.0 25-150
20.0 6-100
1 N,N'-methylenebisacrylamide is included at 1/30th the concentration of acrylamide.

 

Effective Range of Separation of DNA in Agarose Gels

% Agarose (w/v) Efficient Range of Separation of
Linear DNA Molecules (kb)
0.3 5-60
0.6 1-20
0.7 0.8-10
0.9 0.5-7
1.2 0.4-6
1.5 0.2-3
2.0 0.1-2

 

DNA Size Migration of Sample Loading Dyes

Agarose Concentration (%w/v) Xylene Cyanole Bromophenol Blue
0.1-1.5 4-5 kb 400-500 bp
2.0-3.0 (sieving agarose) 750 bp 100 bp
4.0-5.0 (sieving agarose) 125 bp 25 bp

 

Troubleshooting Guide for SDS-PAGE Protein Electrophoresis

Problem Possible causes Solution
Faint or missing protein bands Load quantity is below the detection level of the stain Check the A280 and increase sample concentration
    Use a more sensitive stain (e.g. silver stain)
  Proteins were not fixed in the gel Use a stain which also fixes the proteins
    Use gel fixing solution
  Small peptides (<4 kDa) did not fix in the gel Fix the gel with 5% glutaraldehyde. Rinse the gel well with water before staining
  Proteins are degraded Check the A280 and avoid protease contamination
  Protein ran off the gel Use a higher concentration PAGE gel. See precast gels for recommended gel concentration or use a 4-20% gel if the size is unknown
Film on gel after staining Precipitated Coomassie Blue R Rinse the gel for 15 seconds in methanol and immediately return to water or destain
Poor band resolution Concentration of protein is high Load 10 μg per protein or 100 μg per protein extract
  Age of the gel, due to base catalyzed Order fresh precast gels or cast a fresh gel
  Improper gel concentration See precast gels for recommended gel concentration or use a 4-20% gel if the size is unknown
Band smearing High salt concentrations Dialyze sample, precipitate the protein with TCA or use desalting columns
  Concentration of protein is high Load 10 μg per protein or 100 μg per protein extract
  Protein aggregation Add 4-8 M urea to the sample
  Voltage is high Electrophorese at 10-15 V/cm
Protein precipitation in the well Hydrophobic proteins Add 4-8 M urea to the sample
White precipitate in sample SDS precipitation Could be due to the presence of guanidine or potassium salts in the sample