![]() Never run a Western blot again Simple Western from ProteinSimple, a Bio-Techne Brand. In most cases, the rest of the troubleshooting issues can be grouped. Observation: High Background Observation: Diffuse Bands Observation: White Bands (ECL method) Observation: Patchy uneven spots all over the blot Tween 20 is a registered trademark of ICI Americas. White (negative) bands on the film when using ECL detection. Check and optimize gel electrophoresis conditions. ![]() Anyway I don't do straight westerns very often but that is my two cents. Since one issue that commonly arises during Western blotting is the presence of unusual or unexpected bands on the blot, our troubleshooting section begins with a table describing some common reasons and potential solutions for addressing and evaluating this type of problem. Carefully remove air bubbles between the gel and the membrane before protein transfer. do you have some reason to expect crisp bands (another persons gel etc) because you will not or will rarely see nice crisp bands like you see in DNA gels when doing western blotting. The gels pore size is not correct for the proteins that need to be separated. I observed band of target protein in SDS-PAGE. Is this by chance a glycosylated or phosphorylated protein? You may expect to see a slightly diffuse band when you have modified proteins like those, so I say it looks good, maybe reduce the amount in lanes 1-2, especially lane 2 (the one just to the left of what i referred to as the marker lane) but the other lanes I think look alot like what you normally see with phosphorylated or glycosylated proteins. Smeared bands in western blot Hello everybody, I have a problem with western blot of my target protein (the size of protein is around 14 KDa). ![]() I really don't think your bands look bad, especially on the right hand side of the gel, and your background is very nice which is also a good sign. So, I may be off base here, but your marker lane (that is what the third lane with bands is right?) looks really good, and I think that by and large this controls for issues with running the gel and transfers.
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