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SDS PAGE - Band artifacts when protein buffer is pH 5.0 and heated

SDS-PAGE band artifacts low pH Sodium acetate buffer

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#1 anaivan

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Posted 14 December 2012 - 10:01 PM

Hi,
Initially I used to purify my protein in Hepes pH 8.0. For certain reasons, now I am purifying my protein with buffer sodium acetate pH 5.0 as final buffer. I observed several bands in SDS PAGE analysis in the end of gel filtration. I figured they could be artifacts of overheating and ran same samples with time of heating as a variable. Interestingly, as I believed, I did not have any unintended bands when sample was not heated but they started appearing as a function of heating time. Another interesting factor is that same protein with buffer as Hepes 8.0, which was heated for 5 mins, did not have any such artifact bands. Please see the attached picture.

I wonder if protein in sodium acetate pH 5.0 can have such detrimental effects when used with sample loading buffer (which is made with b-mercapto ethonol along with usual components). I welcome any inputs explaining/suggesting solution to my problem. Thanks in advance. (I Hope asking questions during weekend is not a bad idea.)

Note: This is a his-tagged protein and Water bath temp was 99 C, if it helps. Those bands cannot be complexing with my protein as band for non-heated sample looks same as heated sample. Sorry for poor quality picture. My lab camera is broke!

Thanks,
Anaivan

Attached Thumbnails

  • Gel_ heating.png

Edited by anaivan, 14 December 2012 - 10:04 PM.


#2 mdfenko

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Posted 17 December 2012 - 07:12 AM

you may have some contamination with an acid protease (eg cathepsins) with your purified protein (may be from some organism living in the protein solution).

maybe acid hydrolysis.
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#3 anaivan

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Posted 20 December 2012 - 12:19 AM

Thanks for your input, mdfenko. This may not be acid hydrolysis as the unintended bands appear only when they are boiled. Otherwise it remains intact.





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