I have extracted my protein using isopropanol from Tri-reagent LS extracted cell culture. I found the pellet is difficult to dissolve in 1% SDS. I have read through some articles saying that adding DTT is helping to dissolve the protein pellet. May I know the final concentration of DTT to dissolve the protein pellet and detailed protocol to dissolve protein in 1% SDS with DTT?
You can use from 50 to 100 mM DTT, but maybe better will be low chaotrope extraction buffer: 50 mM Tris·Cl, pH 7.0, 5 mM EDTA, 3 M guanidine·HCl, 10mM DTT. You can also add SDS or increase the concentration of guanidine·HCl.
I am not sure about the DTT concentration. What I am talking here is my personnel experience. I have also faced this kind of situation when I usually dried the proteins extensively in the vacuum dryer. If possible avoid extensive vacuum drying. I could dissolve the same amount of protein in 1% SDS with perfect vacuum drying which I could not dissolve in 1% SDS previously with extensive vacuum drying.
Perfect vacuum drying is just enough drying to get rid of the solution, don't allow your protein to become powder in vacuum dryer.
Thanks Jaroslaw for the suggestion. I will try out.
Thank you Manoj. I only air dried for 5 minutes, so I don't think it is over dried. Anyhow, thanks for the suggestion.
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