cells are lysed in RIPA buffer and I want to use that extract for IP, As extract has NP-40, SDS, high salts it will interfere with the binding of Ab with desired protein. Dialysis is one method but can anyone tell me if its possible to pellet down soluble protein extract only by centrifugation?
You may try Perchloric acid precipitation, centrifugation and neutralisation with KCl. But I am not sure if all protein precipitates and if all soluble proteins recover resonably. I hope other collegues have better ideas...
TCA or Trichloroacetic precipitation is a similar approach to what Erik suggested. Similar caveats.
If you need a free- salts protein preparation, you can use TCA precipitation followed by cold Acetone precipitation. This way you can remove residual salts. Watch this protocol and hopefully useful for you.
TCA Precipitation Protocol
1. Add an equal volume of 20% TCA (trichloroacetic acid) to protein sample.
2. Incubate 30 min on ice.
3. Spin in microfuge at 4 deg. For 15 min.
4. Carefully remove all supernatant.
5. Add ~300 ul cold acetone and spin 5 min at 4 degrees.
6. Remove supernatant and dry pellet. You can do steps 5 and 6 twice to obtain better results.
7. Resuspend samples in desired buffer.
Warning: TCA is a strong acid and should be handled with care
First to answer the original question: No! There is no way to spin down your proteins from solution!
So try the TCA, in our hands we precool the 20% TCA on ice, than the precipitation is faster. Sometimes afterwrds it is difficult to resolve the pellet again in the right buffer. So, give it a try!
Oliver, strictly speaking, it is incorrect that there is no way to spin down proteins from solution; sedimentation coefficients of proteins are determined by watching them move under centrifugal force - centrifugal force works, it's just a matter of how much and how long! ;)
It may not be, on the other hand, the most practical way to get the job done.
Selection of a suitable method depends on the nature of target protein(s) (are they soluble proteins, sensitive to changes in pH, membrane bound etc.), their final use and their stability under conditions of recovery method employed.
If the protein(s) of interest are highly lipophilic (lipoproteins), they may or may not completely solubilize after acid precipitation and in some cases may not easily or completely precipitate by conc. Ammonium Sulfate.
Without knowing enough about your target protein(s), suggestions can only be general and perhaps even less than useful.
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