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Protein resuspension and 1d separation

Protein resuspension and 1d separation - Protein Forum

Protein resuspension and 1d separation - Protein Forum


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  #1  
Old 06-28-2011, 03:18 PM
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Default Protein resuspension and 1d separation



Greetings,

I am having a problem running acetone precipitated protein on an SDS-PAGE gel. This is protein precipitated from bacterial culture supernatant. Using acetone, it appears to yield a visible protein pellet. However, I am having trouble getting this sample, after re-suspension in sample buffer (laemlli), getting any protein to show up on my SDS-PAGE gel. I am boiling the protein samples. My bradford assay indicates that there is roughly 2ug/ul in the sample. I am centrifuging after boiling to clear any aggregates or non suspended proteins.

When I stain the gel with CBB, it looks like a deep blue band on the bottom of the stacking gel, indicating that perhaps the protein never actually entered. My ladder runs fine, so I doubt that the gel itself is the problem.

Does anyone have experience with sample precipitation utilizing acetone? Most of my protocols say that I shouldn't dry the pellet too much, others indicate that it should be lyophilized before use. Seemling contradictory. Anywho, any insights would be appreciated.
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Old 08-20-2011, 05:42 AM
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Default Re: Protein resuspension and 1d separation

I've done plant protein extraction using acetone before and I usually air dry my pellets to completely dry form without any problems. I use 12% resolving and 5% stacking SDS-PAGE. Perhaps you may want to change the re-solubilizing buffer, the common one will be 1x SDS sample loading buffer- 50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA and 0.02 % bromophenol blue. However, the buffer is not compatible to Bradford assay.
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Old 09-21-2011, 08:41 PM
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Default Re: Protein resuspension and 1d separation

Thanks, I'll give this a shot. I don't think I've completely dried the sample out, due to fears of infinite failure
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acetone precipitation , protein , protein pellet , resuspension , sds page , separation , supernatant protein


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