
Post Electrophoretic Analysis
Faint Bands, High Background
A high background that obscures the bands, or in combination with fainter than usual bands, indicates dye binding to the gel matrix, or contamination of the matrix with a dye-binding material (most often a protein).
Coomassie Blue R-250:
- Destaining time too short: It takes hours for the dye to completely diffuse out of the gel after thorough staining. Early in the de-staining process, the background will be high and the bands weak. As the destaining progresses, bands will intensify somewhat as they capture free dye in the gel, and the background will decrease as dye elutes from the gel.

ProtoBlue Safe/Colloidal Coomassie:

SDS in the gel: The primary cause of high background with Protoblue Safe is the presence of SDS in the gel. If the preliminary washes are not carried out

properly, enough SDS will be carried into the staining solution to disrupt the colloidal dye suspension. As the colloidal particles break down, they allow the dye to diffuse into the gel. This background will eventually dissipate, but it can take overnight or longer to get a clear background. SDS will also inhibit the binding of the dye to the protein bands, further compromising sensitivity. Always use the completed pre-washing protocol.Protein contamination of the gel: As with R-250 above, gels can be contaminated with protein if the reagents are old or impure, or if a sample of high protein concentration is allowed to diffuse into the upper running buffer. Use fresh, high-quality gel and buffer reagents, and load only small volumes of high concentration samples.
- UV Shadowing
- Uneven Staining
- Staining Proteins Immobilized on Membranes
- Staining Protein Gels with Coomassie Blue
- Southern Blotting
- Smeared Bands
- Silver Staining Protein Gels
- Silver Staining DNA Gels
- Protein Fixation on Gels
- Post-Electrophoretic Visualization with Nuclistain
- Overview of Western Blotting
- Northern Blotting
- Method for Western Blotting
- Mechanism of Immunostaining
- Mechanism of Immunostaining
- Immunostaining with Alkaline Phosphatase
- Guide Strip Technique
- Faint bands, low background
- Faint Bands, High Background
- Ethidium Bromide Staining
- Enzyme Linked Immunosorbent Assay (ELISA)
- Coomassie Blue Stain- Troubleshooting
- Blotches on Gel
- Autoradiography
- Autoradiographic Enhancement with Autofluor
- An Overview of Northern and Southern Blotting
- Alkaline Blotting