Hi,
I am running an experiment in which I use acetone precipitation to concentrate my supernatant. I then resuspend the pellet in a solution of 1X sample buffer in PBS, run SDS-PAGE, and then Western blot. Unfortunately, I am consistently seeing band distortion / smearing. My gel front is uneven, and the ladder expands into the surrounding wells. When I use a Ponceau stain after transferring, there is a large smear in the upper half of the gel (photo attached). What might be causing this? How can I try to address this issue? I don't think it's residual acetone, as I have heat-dried my samples and lyophilized for good measure.
Thanks to anyone that can help!
Typically looks like there's leftover acetone but I'd suggest
i) what is the original buffer prior to acetone ppt? acetone can co-precipitate salt at high enough concentration because what you're doing is 'dehydrating' the proteins so that the hydrophobicity forces precipitation. But if you've got plenty of salt in the original buffer that can also precipitate, and when you resuspend in 1x SDS sample buffer in a lower volume - you'll actually concentrate the salt more.
ii) perhaps after resuspending after acetone ppt with SDS buffer, spin it hard and take off the supernatant and load that fraction on a gel.
The problem seems to be that the protein aggregation is so severe that the electric field has been distorted. Have you tried *not* heat-drying your samples? Acetone can promote protein crosslinking (Ref), especially with heating. In fact, I would keep the entire acetone precipitation process cold, and remove acetone through lyophilization only. Please keep us posted on how it goes. Good luck!
Ref. https://www.liverpool.ac.uk/pfg/CPR_Pubs/files/97695eb8569585029689d7dbc93bf292-10.html)
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