What I was testing was whether my suspected antimicrobial agent can cause cell leakage, so I incubate the cells in the PBS solutions with my chemicals in. After incubation, I centrifuged the cell and get the supernatant, then I used TCA to precipitate the possible protein from the supernatant. The Bradford assay was done after I suspended the precipitated pellet in 1M Tris pH8.8. Also the spectrophotometry indicated there were significant higher absorbance at 260&280 nm in the supernatent in the treated samples. So I believe there were things leaking out.
I solubilized the pellet by neutralization for electrophoresis, In some of the treated samples, the proteins shows up in SDS-PAGE. However, some of the treated samples still not show up after neutralization, I was thinking is it possible that some cytoplasmic protein cannot precipitated by TCA? Or the protein concentrations were not enough to be show up as bands (but Bradford assay indicated the protein content was 10ug/uL)?
Thank you!
Hi Kangzi
If Coomassie staining was used then rinse the gel very well with methanol only and add silver stain which is more sensitive than Coomassie
Hope this helps
Hi Kangzi,
After TCA precipitation and addition of SDS sample buffer, make sure that in your samples, the bromophenol blue indicator is blue. Sometimes, a trace amount of TCA may upset the final pH of the sample buffer in which case, the indicator dye will be yellow color. If you run the sample with this pH, proteins will not be resolved..
Good luck,
Hediye.
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