The bands from the far-western are white, why is that? How can this be solved?
The appearance of white bands in Far-Western blot experiments is usually related to exposure duration or issues with the detection system. Here are some possible causes and solutions:
1. Overexposure
Cause: Too long exposure time can lead to excessive signal enhancement, causing bands to 'invert' and appear white instead of black.
Solution: Shorten the exposure time or reduce the amount of developing solution (such as substrate or chemiluminescent reagent).
2. Excessive protein
Cause: The concentration of protein in the sample may be too high, leading to saturation of the detection system and resulting in white bands.
Solution: Reduce the amount of protein sample loaded, or re-optimize the protein transfer step.
3. High antibody concentration
Cause: High concentration of primary or secondary antibodies can cause high background or even inversion of colors.
Solution: Lower the concentration of antibodies, and perform gradient dilution experiments to optimize antibody concentration.
4. Substrate issues
Cause: Abnormal reaction of the substrate, such as uneven distribution of chemiluminescent substrate, may cause the band area to appear white.
Solution: Ensure even distribution of the substrate and use fresh substrate. Replace the detection substrate if necessary.
5. Membrane processing issues
Cause: Uneven or incomplete protein transfer on PVDF or NC membranes may lead to areas without protein signals, resulting in white bands.
Solution: Check the protein transfer step, ensure electrophoretic transfer conditions are appropriate, and use stains (such as Ponceau S) to verify protein transfer efficiency.
Try these solutions and make adjustments during the experiment to observe improvements in the bands.
Biotyech - A leading service provider for bioproduct characterization and multi-omics mass spectrometry analysis.
Related services:
Crosslinking Protein Interaction Analysis
SILAC combined with Immunoprecipitation and Mass Spectrometry for Protein Interaction Analysis
How to order?
