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Looking for precipitation method to reduce the volume of my protein complex elu


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#1 AllenChiu

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Posted 09 September 2010 - 11:30 PM

I want to reduce the volume of my eluate of protein complex by precipitation and resloving in small volume so I can load them into SDS-PAGE gel.The eluate comes from the elution of Strep-Tactin Superflow resin with elution buffer with 2.5mM D-Desthiobiotin in it.
The precipitation method should be able to precipitate each proteins in the former complex no matter the abundance is low or high.
The precipitation should be able to readily been resolved again without changing the profile of former composition of proteins.
Then what is your suggestion? As the TCA precipitation is hard to be resolved again.

PS:My overall purpose is to discover new interacting protein of protein of interest by Strep-Flag tandem purification.

#2 mdfenko

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Posted 10 September 2010 - 07:32 AM

if your only purpose is to load onto sds page then tca precipitation is your best bet.

after centrifuging wash the pellet one or two times with acetone to remove tca. the pellet should dissolve in sds sample buffer.
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#3 AllenChiu

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Posted 10 September 2010 - 07:37 AM

if your only purpose is to load onto sds page then tca precipitation is your best bet.

after centrifuging wash the pellet one or two times with acetone to remove tca. the pellet should dissolve in sds sample buffer.

It seems the precipitation will be more easily dissolved in the absence of TCA?

#4 mdfenko

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Posted 10 September 2010 - 07:49 AM

It seems the precipitation will be more easily dissolved in the absence of TCA?


possibly, but you remove residual tca so that it doesn't affect the sample buffer. if you don't remove it, the sample should still dissolve but your bromphenol blue may turn yellow. then you'll have to neutralize it (we use a few microliters of 0.5M tris pH 9) or it will affect the migration of the sample in the gel.
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#5 rkay447

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Posted 10 September 2010 - 08:11 AM

Rather than precipitate you can always try to concentrate the sample using vivaspin columns. I'm taking a rather large volume (about 2mL) and concentrating it down to 35ul and running everything on one SDS-PAGE gel with very nice results.

#6 AllenChiu

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Posted 10 September 2010 - 08:12 AM

Rather than precipitate you can always try to concentrate the sample using vivaspin columns. I'm taking a rather large volume (about 2mL) and concentrating it down to 35ul and running everything on one SDS-PAGE gel with very nice results.

will it take a long time?

#7 rkay447

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Posted 10 September 2010 - 09:26 AM

It depends on your starting volume, the amount of protein present and if you have a detergent in the buffer. At first, when I had NP-40 in the buffer it took about 7 hours of centrifugation but this last time I did the peptide elution and final wash without detergent and it only took about 3 hours. If you have a ton of protein in the sample, that will slow things down as well. Also, there are different columns that have different molecular weight cut offs and I think the lower the cut-off, the longer it takes because you are keeping more protein in your sample. I use the lowest possible cut-off which is 3kDa.




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