Dear All,
I would like to elute protein band from SDS-PAGE gel stained by coomassie brilliant blue r-250 stain. which will be used for biological activity & mass determination. So which of the following buffers are more suitable for that.
1. Use 0.5-1 ml of elution buffer (50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM EDTA; pH 7.5).
2. formic acid/ water/2-propanol (1:3:2 v/v/v) (FWI).
please suggest.
regards.
Hi Mohammed,
when you say biological activity, what are you specifically refering to?
Because running a protein in SDS-page will denaturate your protein, and staining wil fixate it. So depending on what kind of assays you want to make maybe the protocol to follow may be changed.
For mass spec, a volatile buffer is preferable, such as ammonium bicarbonate.
I agree with Edward Valera-Vera concerning attempting to measure biological activity.
Dear Edward Valera-Vetra,
i am running sds- page to detect my protein of interest from the total crude extract especially i am interested in low molecular weight protein from 25 kd and lower, i want to detect its biological activity anticancer, antibacterial and toxicity.
Proteins bands could be directly extracted from SDS PAGE into a solution consisting of Formic acid/water/2-propanol then used for MS analysis alternatively you can excised protein bands detained completely removed the coomassie blue then extracted using the Tris buffer.The biological activity most likely will be lost due to denaturing effect of SDS to avoid this you might use none denaturing gel electrophoresis
good luck
Dear Mohammed, if you think that your protein can refold again after SDS-PAGE or its activity is not affected by the SDS binding I would suggest to use the 1st buffer, because its components are not harmful for downstream applications of the extracted protein. But to remove efficiently SDS that might interfere with protein activity you need to use some alcohol, like methanol or ethanol. 2-Propanol may also help to remove SDS. You may try to use the second buffer to extract protein and then exchange the buffer to the first one using dialysis or by centrifugation in Centricon (or Amicon tubes, Millopore company).
Dear Demitry Karpov,
thank you for your suggestion i too feel to do so, but the second buffer as it have salts and detergent will not harm the activity and the structure of the protein.
regards
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