Hi!
I want to deproteinize a sample and then apply the resulting deproteinized product to cells in culture (PBMCs). Which method can I use for this? I thought about TCA, but I am not sure whether, even after neutralization, the sample would be toxic for the cells.
Thank you in advance.
Dear Dr. Patricia Román-Carrasco
If you have doubts about using TCA or PCA precipitation, which are the most extensively used deproteinization protocols, you could use high temperatures (heat above 50 degree C), provided the sample is not affected by the treatment. Such treatment supply kinetic energy to protein molecules, causing their atoms to vibrate more rapidly and disrupting relatively weak hydrogen bonding and dispersion forces.
Alternatively, you could use salt induced precipitation with ammonium sulfate. The process of “salting out” a protein can occur with a number of different types of neutral salts, but ammonium sulfate is by far the most common. With salting out, the protein interacts with the salt as opposed to the water, which leads to less interaction between the water and the protein’s solvation layer, which in turn leads to more hydrophobic patches being exposed and to encourage those patches to interact with one another. This causes proteins to precipitate.
Ammonium sulfate is preferred because it has a high solubility rate. You may either add it to the protein solution, or the solution may be poured onto a measured amount of ammonium sulfate.
The protocol in brief is as follows.
1. Transfer sample to a beaker containing a stir bar and place on a magnetic stirrer.
2. While the sample is stirring, slowly add saturated ammonium sulfate to bring the final concentration to 50% saturation. Please note that,
a. Volume of ammonium sulfate needed is equal to the volume of sample.
b. Adding the ammonium sulfate very slowly ensures that local concentration around the site of addition does not exceed the desired salt concentration.
3. Once the total volume of ammonium sulfate is added, move the beaker to 4°C for 6 hours or overnight.
4. Transfer to conical tube and centrifuge the precipitate at 3000g for 30 minutes.
5. Carefully remove the supernatant without disturbing the pellet.
6. Use the supernatant on cells in culture.
Regards,
Malcolm Nobre
You could use an Amicon® Ultra filter device. There are two main types, one uses high pressure nitrogen to drive the solution through the filter, the other uses centrigugal force. The lowest molecular weight filter offered has a nominal molecular weight limits cutoff of 3 kDa. If the proteins you are trying to remove have molecular weights greater than ~3 KDa (that's equivalent to a peptide of about 27 amino acids) this method would require the addition of no precipitants.
Thanks a lot for the answers! Philip G Penketh I also thought about Amicon® Ultra filter devices, but my doubt in this case was whether I would be able to preserve the lipids and carbohydrates while deproteinizing the sample.
Patricia Román-Carrasco, since it is based on pore size I would expect it to preserve most lipids and carbohydrates unless they were large polymers such as glycogen etc.
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