I run western blot on HCT-116 cell lines, I got actin band like in the attached picture. The band size is not same. Can anyone help me explain this situation and how to overcome? Thank you your response!
Can you please give some more details? Western blotting has many step and problem(s) can occur in each step. Are these bands belong to different membranes? Band intensities seem more or less similar in actin bands but the bottom one seems brighter.
Difference in band size may occur due to loading error or protein preparation process. On the other hand, fresh primary and/or secondary antibody results in brighter bands. It seems saturated.
If you can give more details, I may help
Good luck
Hi Jenny Bao Tran Le
I would first measure your protein concentration using BCA or Bradford assay. Then load the same amount of protein into your wells and run that. That should equalise your bands.
Like Miray Turk said about the saturation of the bands, the lower actin seems to be saturated. In my opinion, you should dilute primary and secondary antibodies to get more refined bands and/or use less protein to start with.
Other than that prior to loading you should carry out Bradford or BCA assay to measure protein concentration in your samples.
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