I am writing a report and I am supposed to determine the purity of the protein sample? Samples were ran on SDS-PAGE and stained with CBB. Using the Rf value and plotting a standard curve, I was able to determine the molecular weight of the protein. However, I am required to determine the purity of the protein. Please any ideas ir suggestions on how to go about this?
if the staining is faint and absorbance still obeying Beers law then you could scan then calculate the area under all of the peaks and a comparison of the total area and the area under the protein of interest absorbance peak will give an approximate estimate of purity bearing in mind that some material may not have stained at all depending on their nature and the staining dye used
I think SDS-PAGE only give rough estimation about purity of the protein. If you have separated you protein of interest and then run it on the SD-PAGE, ideally you should get one band which indicate that the you have successfully purified the protein but as SDS-PAGE separate protein on one dimension, there are chances that single band may comprises of more more then one type of protein. You can either go for 2D gel electrophoresis or if your protein is enzyme you can find specific activity which is highest when protein is in its pure form.
Or further you can check the protein purity on LC-MS/MS which is very sensitive technique.
Thanks for your input Pooja Sevak But all i have now is the picture of the gel, the lab session is over and there's currently no way I can do LC/MS/MS. I am really stuck right now.
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