Currently I am working on solubilizing the ATP/ADP antiporter NTT1 (or AATP1) from Arabidopsis. We expressed the WT gene with optimized codon usage in yeast and solubilize the membrane pellet in the presence of 1% (m/v) LAPAO, according to the protocol of Deniaud et al. (DOI: 10.1371/journal.pone.0032325) .
Sadly, I was not able to make a single acceptable SDS-PAGE gel from my solubilizations. Blots of non-solubilized protein are ok. There is one thick band at the top and that's it. Tried denaturation at 99°C, lower temperatures, let the extract with sample buffer stay over night, add more sample buffer, doesn't matter, western-blot example attatched (protein is expected between the red line and the blue line below, temperatures between 30°C and 99°C). I can imagine that non-ionic and amphotere detergents may strongly block the proteins surface against SDS, so only a small amount will end up with a negative netto-charge and will be separated. The reference paper shows SDS-PAGE gels as well, they look fine, but they didn't mention their SDS-PAGE protocol.
Does anyone have any advice how to cope with this problem? Thank you!
It's possible, I suppose, that the amphoteric detergent is interfering with the electrophoresis. Try precipitating the protein extract with trichloroacetic acid to get rid of the amphoteric detergent. Re-dissolve the pellet in SDS-PAGE sample buffer.
It's possible, I suppose, that the amphoteric detergent is interfering with the electrophoresis. Try precipitating the protein extract with trichloroacetic acid to get rid of the amphoteric detergent. Re-dissolve the pellet in SDS-PAGE sample buffer.
Agree with Adam, you may need to add a bit of 1M TrisHCl pH 8.5 to counter any residual TCA.
Hi Andre, I agree with the above comments, but in lieu of TCA precipitation, you could try sonicating the sample in the presence of 1% SDS.
Alternatively, you could try removing the detergent using:
https://tools.lifetechnologies.com/content/sfs/brochures/TR0019-Remove-detergent.pdf
Thank you for your comments. I tried TCA precipitation (10% v/v TCA in the solution) for 10 minutes at room temperature, followed by 12 minutes centrifugation at 10.000 g and washing the visible pellet with ice cold 80% (v/v) acetone. Silver stain says no. Maybe I wasn't careful enough at the washing step, I'll give it another try this week.
For a first and fast solution (trial) the TCA precipitation protocol is worth for trying. In case of nonionic (or amphoteric) detergents the protocol for removing detergents and lipids published by Wessel and Flügge is recommended (Anal. Biochem. 138 (1984) 141-143.). This protocol is tedious (time-consuming) but results in excellent sample for SDS-PAGE.
Hi what about acetone precipitation, it covers better the whole spectrum of proteins.
You can use as well this kit:http://www.merckmillipore.com/PL/pl/product/ProteoExtract-Protein-Precipitation-Kit,EMD_BIO-539180#anchor_Product%20Information
cheers
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