Does your protein have a tag that Magnetic Beads or Agarose Resins can bind to?

Sebastian Lühn hello, our protein currently do not have a tag so magnetic beads or agarose resins will not be able to bind it.

Maybe a specialized protein resin/MagBeads can be used here.

Hopefully this gives some inspiration.

https://cube-biotech.com/customized-resins-maximum-specificity

The TCA/acetone pellet may well be very small and glass like so not white. Be careful decanting supt from ppt as not to dislodge pellet. Check protein content of media prior to ppt to give you a rough estimate of total protein present.

Ammonium sulphate ppt could be used effectively. Proteins will ppt at different percentages, but 70%+ should do it for most proteins but should be tested.

Agree with Ian above. Not sure what your intentions are with your precipitate, but if you intend to perform any kind of biological work on the protein, TCA/Acetone is unlikely to be compatible anyway. Ammonium sulphate is the way to go (you may have to go higher than 70% if your culture media is dilute; try 70-90% range). Other option since you're likely to have a fairly large volume is various readily available affinity procedures (ion exchange etc..). The approach used will depend on your aim (analytical vs preparative).

Were you trying to precipitate proteins from bacterial extract or were you trying to precipitate proteins from the culture medium? If you were trying to precipitate proteins from bacterial extract, it is possible that the method you used to break open the cells and extract the proteins did not work. You could test this by determining the protein concentration of the solution to which you added the TCA/acetone. If the concentration is very low, you could try a different method of extracting proteins from the bacteria. If you were trying to precipitate proteins from culture medium, it is very possible that the solution is too dilute for TCA-acetone precipitation to work. You might need to concentrate the proteins using another method such as ultrafiltration. Depending upon the volume of supernatant that you need to work on to get enough protein to show up on a Western blot, you could use ultrafiltration spin columns or ultrafiltration stirred cells. If you need to work on a large volume of culture medium because your protein of interest is in low concentration, you may need to start with a large spin column or stirred cell, then re-do the ultrafiltration on a spin column or stirred cell of smaller size. It is also good to remember that sometimes proteins stick to the ultrafiltration membrane. So, after retrieving the concentrated solution, add a small volume of fresh buffer to the membrane, leave the buffer there for 15 minutes or stir gently if you are using a stirred cell, then retrieve that buffer.

Sébastien Soubeyrand Hello. Thank you for your answer. The precipitate was meant for western blot to quantify the amount of protein our bacteria secreted. I will give ammonium sulphate a try then in this case.

Maybe I am thinking too simple, but if you only want to know if and how much of your protein is made you can just take a sample of your sup and analyse that on SDS-page/western blot directly?

For the rest I agree with Sébastien Soubeyrand and the others. It depends what you want to do with the protein.

Hi, Alicia Tan and all others.

I do agree with Anna and would only ask you If you tested your sample by performing an SDS-PAGE stained by coomassie. If so, Did your bacteria produced secreted, soluble or insoluble protein? Did you add a secretion marker to your heterologous protein sequence? Is it a recombinant one?

So, If you could see your protein at this SDS-PAGE step you should be able to find out easily. If not (Or If it is not at the secreted medium), maybe it is not being secreted or your lysis method is not working.

All the best.