Why does my Western blot look dirty?
Blotchy, Flecked, Or Dirty Background These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer.
What causes band smearing?
If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel. In addition, if the gel is moved after the sample is placed in the well, it can cause the sample to spill out of the well. This can also cause smearing.
What causes smear?
Smearing also results from poor sample quality. For example, a DNA sample contaminated with protein or containing too much salt may produce smearing. Degraded or denatured samples also yield poor results, including smeared bands.
What causes streaking in SDS PAGE?
Sample preparation problems. The most common cause of horizontal streaking is a problem with sample preparation. Any contaminant with a net ionic charge will affect isoelectric focusing and lead to horizontal streaking. Nucleic acid contamination of samples can also contribute to horizontal streaking.
What do the bands mean on Western blot?
Western blot is often used in research to separate and identify proteins. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present.
Why are my bands smeared in gel electrophoresis?
1. Improperly prepared gel: If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear. If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel.
What does the smearing indicate?
Smearing indicates degradation of the genomic DNA. Since the 1 kb ladder is sharp and fine, the degradation could have happened during extraction process and not during electrophoresis.
Why do I have bands on my Western blot?
Western Blot / WB is used to detect specific proteins in the given samples. Sometimes, smeared bands may appear in a Western Blot / WB, this makes it difficult for you to figure out which is your protein of interest.
What’s the maximum sample size for Western blot?
Request a free Western blot tips, tricks and troubleshooting guide. Reduce the sample loads. The maximum recommended sample load for optimal resolution in mini gels with 10, 12, 15, or 17 wells is 0.5 μg per band or about 10–15 μg of cell lysate per lane.
Why do I have multiple bands on my protein blot?
However, analysis of the protein can be difficult if multiple bands appear on the blot. When dealing with multiple bands on Western blots, it is important to determine whether they are due to technical artifacts or whether they represent true variants of the protein of interest.
What can western blot assay tell us about a protein?
The Western blot assay provides valuable information about a protein including abundance, the apparent molecular mass, post-translational modifications and splice variants.