Hi all,
I want to characterize a product-related impurity band appearing in SDS-PAGE analysis. I excised the band and incubated it in elution buffer containing 0.1% SDS. Now I want to analyze the eluted impurity using a reversed-phase column (Zorbax 300SB-C3), because the peak profiles of my protein of interest in RP-UPLC is fairly well understood. But I think SDS in eluate would adversely affect the LC analysis. How can I effectively remove this detergent from the eluate? I tried amicon from Millipore for this purpose, but it barely worked (as opposed to the manufacturer's claim). I also understand that precipitation with acetone is one of the most effective ways of removing SDS from proteins samples, but in my experiences, re-dissolving the resulting pellet was technically challenging without the aid of, again, SDS.
Any comments would be greatly appreciated.
Here is a paper offering a possible solution to this problem:
https://pubmed.ncbi.nlm.nih.gov/2363498/
Dear Dooyoung Lee ,
These two references I found, as suggested in the following question:
https://www.researchgate.net/post/How_to_elute_a_protein_band_of_interest_from_SDS-PAGE_or_NATIVE-PAGE_gel
They are:
Castellanos-Serra LR, Fernandez-Patron C, Hardy E, Santana H, Huerta V. High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level. Application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins. J Protein Chem. 1997 Jul;16(5):415-9. doi: 10.1023/a:1026340923032.
https://pubmed.ncbi.nlm.nih.gov/9246622/
Kurien B.T., Scofield R.H. (2012) Extraction of Proteins from Gels: A Brief Review. In: Kurien B., Scofield R. (eds) Protein Electrophoresis. Methods in Molecular Biology (Methods and Protocols), vol 869. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-821-4_33
Best regards.
Passing the eluted protein containing solution through a small Sephadex G-10 column may remove the SDS. You may pack Sephadex G-10 in a 1 ml insulin syringe barel. This shall work!
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