In my experience, precipitating a protein normally deactivates it. In my experience this is primarily due to the fact your protein is already "precipitated", that is, it is in inclusion bodies, which are always in the insoluble fraction of your cell lysate. Extracting protein out of inclusion bodies is a difficult task when trying to maintain protein integrity and functionality, since denaturation is often required. The procedure that is often used here is to lyse your cells in a denaturing buffer. One usually experiments with typical agents like: 8M Urea, 6M Guanidinium chloride, 10% SDS or 10% Tween. Always have a reducing agent as well, 5 mM BME or TCEP usually works. If you have other denaturants/chaotropics or reducting agents, it is good to give them a try. I worked with a rather stubborn protein that did the same thing, nothing in the soluble fraction after cell lysis. Turned out it could only be solubilized in 10% SDS, arguably one of the harshest of the above denaturants. The tricky part will be buffer exchange to refold your protein after isolation. If its current absence from your soluble fraction is any indication, you have a lot of work to do to get it soluble, stable, and functional. Many proteins do not reconstitute after precipitation, so I don't think it is the best idea, unless you already know it has good reconstitution potential. Precipitation usually does not maintain functionality, as precipitation often refers to a disorganized non-solubilized state, compared to crystallization, which can potentially maintain native folding. However, crystallization of proteins (as you must know), is an especially difficult process with some proteins, and can take years of experimentation and observation to complete.

Sorry but I didn't get all the details. By supernatant are you referring to the culturing medium or to the soluble fraction of the cell lysate as suggested y Jordan? I understood the first, if this is the case:

1) The cells may die, but the reason why you see the highest levels of o/e at 48hrs post transfection and then a decrease is that the amount of plasmid per cell lowers with time after a transient transfection. You may want to extract protein at 48 and 72 hrs only for high yeld. 

2) If you are trying to purify a protein from the supernatant I guess it should be a secreted one. Trivial question, is this the case for your protein? It seems that you get sufficient amount of o/e to detect it in cell lysates. If your protein should be secreted, and the plasmid sequence is ok (all the protein domain necessary for secretion are there) then it could be a problem of concentration, don't expect high recovery in the supernatant. You could pull and concentrate supernatants from many plates (better if you generate stable cell lines overexpressing your protein) to obtain a detectable amount of it (regarding the activity I am no expert so I cannot suggest you anything, it really depends on the protein folding stability). 

Good luck

I agree with Francesco: Either your protein is not secreted into the culture medium or  if it is secreted it is below the limit of detection. First you need to be sure that the protein is secreted and this can probably be done by employing more sensitive assays such as ELISA. Concentrating the culture medium is another good idea. 

Hi,

If you want to precipitate your active protein, and the protein is still in the cells, just lyse the cells in a suitable buffer and low detergent concentrations. After clearing the unsoluble proteins by centrifugation, you can then immuno-precipitate your protein of interest (if you have an antibody against your protein). if you don't have an antibody for your protein that works in immunoprecipitation, clone an immunoreactiev epitope in front of  your cDNA (like a Flag-tag, GST-tag or HA-tag). If you use chemical-based precipitation (acids, isopropanol or other) as suggested above, your protein will be inactivated and you need to refold it (as mentionaed above).

We have purified GST-tagged kinases from Sf9 insect cells by mild detergent lysis. The protein was not secreted, it was all in the cells. So we lysed the cells in PBS+0.2% NP-40+protease inhibitors (10 min on ice). Then cleared the lysate (10 min at 10000xg, 4°C), and used the lysate for GST-pulldown (using glutathion-sepharose): overnight incubation at 4°C on turning wheel. The next day, we washed the sepharose-beads 5 times in lysis buffer (same volume as the original lysate), and eluted the protein with reduced Glutathion. If necessary, you can dialyze the eluted proteins, to remove the GST.

A final note: you will need much more than a 24 well plate to immunoprecipitate active proteins. You need at least a 60cm² dish, and much more if you want to purify  µg or mg of protein. But I assume you used the 24 well plates just to analyze expression pattterns.

Good luck!

Hello, Everybody,

Thank you very much for your time and for your detailed suggestions. 

The supernatant I am calling is the culture medium. I collected the culture medium and centrifuged at 12000g for 10min to get rid of the dead cells and cell debris, then I kept the supernatant and pellet seperately to purify the protein of interest. Starting from here I want to precipitate the protein of interest from the supernatant. 

The protein I am studying is a secreted protein. We have already confirmed that with Elisa. I used 24 well to analyze expression patterns, after that I scalled up to T25 flask. 

Hi Burkitkan,

If you can detect the secreted protein by Elisa but not by WB, then I agree with Francesco and Mohan: probably the sensitivity of WB is too low. I'd try to load more of the supernatant on WB (or concentrate it first by using protein concentration filters/columns). You can also try to immuno-precipitate your protein from the supernatant first, and load this on gel.

But if the majority of the protein remains in the cell, and secretion is not required for its activity, you may consider to purify your protein from the cells, rather than from the supernatant.

good luck,

sasker

Hi Sasker,

I did tried with more of the supernatant, but the result is the same. I think I will concentrate it by using a concentrator containing a cellulose membrane with molecular weight cut-ff 20k. Do you think which protocol is better? or Can salt it out with ammonium sulfate? 

If purify my protein from the cells, I think there will be thousands of other proteins and it will be challenging to get a pure protein. 

Thank you for your time

Hi Burkitkan,

That is true, you wil have more background-proteins in a cell lysate.They can be removed with proper washing, elution and perhaps size-exclusion chromatography afterwards - but it may indeed be more challenging.

I have no experience with ammonium precipitation. If it involves protein denaturation, I wouldn't do it.

The cellulose membrane filter should should be the fastest option, if you have enough protein secreted. I have done this before preparative BN-PAGE, and it works. You can give it a try on a small scale format. But the cellls secrete other proteins as well (and if you use serum in the medium, these serum proteins are concentrated as well), so you will need to do an additional affinity purification  (antibody-based or tag-based) or size-exclusion filtration to get your active protein purified (same affinity purification is required for ammonium sulfate precipitation).

good luck,

sasker

Hi Sasker,

Thank you very much. As you said, I will try the cellulose membrane filter on a small format. If it doesn't work, I will try the ammonium sulfate precipitation method. By the way, I am using serum free medium. 

Your suggestions helped me a lot. 

Dear Amita, 

Thank you very much. Does concentrator help or not?