checking protein purity in SDS-PAGE - evaluating the protein purity analysing the 1D SDS-PAGE (May/19/2008 )
Hi everybody, i would like to know some questions about checking the protein purity in a SDS-PAGE gel:
1. First of all, if i wanted to see whether my protein is pure or not, how much protein in microgram quantities should i load in the well? i am running 20 ug of total protein and see many bands... is this right? or should i load 1 ug instead? in this case, contaminants will decrease and i will apparently have a 'more' pure protein in the lane... which one is the best choice?
2. Is there any research paper, reference, website, university, book, etc. that could help me to get a better scope on this?
thank you very much,
Can you elaborate upon what you are checking? Is it an affinity purified (GST, His etc) protein or a chromatography, gradeint, or a lysate with over-expression?
In case of purified ones, you want to have as less other bands as possible, and should also make sure that there are no degradation products. If you are using Coomassie staining, your detection sensitivity is about 100 ng, so 1-20 ug anything is fine. You can actually load 100ng, 1 ug, 10 ug in separate lanes. This will not only give you a choice to select teh best photograph, it will also kind of quantify it. Run BSA lanes for quantification and GST lanes for degradation.
Check a title of your choice here, and have a happy week
Another alternative is silver staining. Lower limit of detection, but fiddly protocol.