In SDS-PAGE, Each lane was loaded with about 100μg of protein. The protein content was determined by the BCA method. It is clear that there is much difference among each lane. who can give me detail explanation.
several questions:
1) 100 ug total protein from cell lysate or 100 ug purified protein?
2) what size of gel
The BCA assay can be affected by any number of interfering substances: http://www.ncbi.nlm.nih.gov/pubmed/23911526
Try the Bradford/Coomassie Plus method or dilute your sample to bring interfering substances below critical levels.
Good luck
100ug protein is a mixture from the fungus culture supernatant, the gel size was mini gel about 9cm x 11cm.
If it is on the gel then you need to think about whether [1] you have large amount of reducing substance [2] your spectro had wrong wavelength (it happen to my fellows several times) [3] Your treatment actually made big changes in proteomics. If the difference in on the membrane then consider your transfer technique.
Make sure that when you measured the concentrations of all the samples that they were all at concentrations within the dynamic range of the BCA assay.
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