I have liters of cell lysate and I need to separate the recombinant protein from the rest. The volume is very large for the affinity chromatography column. Any other technique? I don't need high purity, I just need a more clear sample containing just protein. I thought about TCA separation but I have concerns about protein functionality afterwords. Does anyone have experience with that?
Use an Akta (or a pump) and a column.
If it has an affinity tag, use the appropriate affinity column to pull the protein out. It will save you a huge amount of time!
Ion exchange would be the next port of call, Q or SP column, elute with a salt gradient. The first pass will likely yield multiple peaks, essentially a partial purification.
Really does depend on what your trying to do, and what you want to do down stream.
Have fun!
TCA precipitation is a bad idea. The protein will be inactivated.
One way to deal with the large volume, if you don't have a pump or an FPLC, is bulk binding. Mix the affinity resin into the lysate. Let it incubate for a while with gentle mixing for a few hours. (Don't use a magnetic stir bar because it will grind up the resin.) Once the binding has happened, pour the whole thing into a column and let the resin settle while draining the column. Then perform the affinity chromatography.
You can also use ion exchange resins the same way to bind up the protein from the bulk lysate, then elute the column with a salt gradient.
Something else you can do is ammonium sulfate precipitation and fractionation. In this method, you gradually add increasing amounts of solid ammonium sulfate while stirring the lysate. After each addition and an incubation, you centrifuge the sample to pellet any protein that precipitated. This can achieve a small degree of purification and gives you a pellet that is a mixture of protein and ammonium sulfate. Then you dissolve the pellet in a buffer and dialyze it to remove the ammonium sulfate.
To isolate the desired recombinant protein from a large amount of lysate, affinity chromatography in batch mode purification would be a versatile technique to apply as suggested. Otherwise, there are techniques that reduce the amount of protein sample by non-specific and non-denaturing precipitation. However further separation (fractionation or purification) of the recombinant protein is needed. Hence, I prefer to use affinity isolation and dialysis or UF membranes in tandem to get rid of small molecules and purify the POI...
Good luck
You can try ammonium sulfate precipitation - Article Protein Precipitation Using Ammonium Sulfate
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