Hi. Recently, as a mean to increase yield, I added 0.1% of Tx-100 into all of my IEX purification buffer including the sample injected. However, when analyzed on SDS -PAGE, I observed that there was no single band of my POI at the target size but instead as the picture shown; strong band in between 50 and 37kDa and a very faint band at 25kDa (is not visible in pic). FYI, my target protein is 49kDa and the GST tag is 26kDa. Is it possible for TX-100 to cleave my protein?
Triton X-100 detergent itself will not have this effect. However, if the TX-100 you are using is old and has been exposed to air, it may have formed reactive oxygen species that could cause damage to your protein.
My practice is to use Surfact-Amps-100 (Thermo-Fisher Scientific), which is a 10% solution of Triton X-100 stored under inert atmosphere in ampules, 10 ml per ampule. After opening an ampule, I store the unused portion at -80 in aliquots. A cheaper alternative would be to start with a new bottle of TX-100, dilute some of it to 10%, and store aliquots in the freezer.
Adam B Shapiro Thank you so much for your answer!! I was actually using an old Tx-100, pretty sure thats the reason.
Are you using new IEX column? If using old column, is it used before for any protease purification in the past. I think check the samples before and after IEX. I don't think it is becuase of only Tx-100.
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