DNA / Protein Electrophoresis and Troubleshooting Tables

• Effective Range of Separation of DNA in Polyacrylamide Gels
  
• Effective Range of Separation of DNA in Agarose Gels
  
• DNA Size Migration of Sample Loading Dyes
  
• Troubleshooting Guide for SDS-PAGE Protein Electrophoresis

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