This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Protein Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.
Can I avoid precipitating bovine serum albumin (BSA) when enriching my 50-kDa protein? (Thread 22603)
Q I would like to recover a secreted protein from the supernatant of a primary cell culture, which I will use for Western blot. I decided to try a trichloracetic acid (TCA)/ acetone protocol, but I expect problems with BSA. The molecular weight of my protein is 45–50 kDa and my medium contains 10% fetal bovine serum (FBS). Can anyone suggest a method for enriching my desired protein without precipitating BSA? I can't perform ELISA or immunoprecipitation (IP) because of cost.
A Do you have a tag on the protein to facilitate purification? You could try using Blue Sepharose to remove BSA. This web page, www6.gelifesciences.com/APTRIX/upp01077.nsf/Content/Products?OpenDocument&moduleid=15318 &cmpid=bcl17094801, explains the details. You may also try serum-fresh medium for expression.
A I did a quick NCBI search. Two low-cost methods for removing albumin are precipitation with NaCl and ethanol or the use of TCA/solvents.
A You could perform a sequential precipitation with ammonium sulfate at the isoelectric point of your protein.
Q My protein doesn't have a tag. Can you suggest a protocol for sequential precipitation?
A You can find tables for ammonium sulfate concentrations used in precipitation protocols online or in any protein manual. Those resources will list a starting amount of salt to be added to a volume of your ice-cold sample to obtain the first 20%. Then you will increase the salt to 20–40%, then 60%, etc. You can choose any number of intervals. It is important in this type of precipitation to add the salt very slowly. Prior to the 50% fraction, you can add a saturated solution of the sulfate salt, but you will need to increase the volume and use solid salt for the higher concentrations. You will first precipitate big proteins; the small ones will appear at the end when you are using higher concentrations.
A Adjust the pH of the protein solution to the pI of the protein of interest. This should make your protein (and others with similar pIs) insoluble. Centrifuge the solution and the protein should pellet. Then resuspend the pellet in any buffer with a pH that is not the pI. The original buffer usually works.
Q Can I use the pI calculated by a program like ExPASy?
A The theoretical pI can be vastly different from the actual pI.
A You could try Centricon. The filters come in different pore sizes. You will lose some of your protein if you use the 50 kDa size, but the 30 kDa size should work. What is the volume of the supernatant?
A Centricon will not work because it only removes salt and water. The serum protein from FBS will be concentrated along with the protein of interest.
A What about ion exchange columns? BSA should have a negative charge at neutral pH. If your protein is positive, you could separate it by an SP or CM column.
A You should do IP. I understand the concern about cost, but other methods are expensive as well, especially if you use reagents and don't get results. The only reasonable alternative to IP is the Blue albumin removal kit.
Q With IP, can I concentrate 50 mL of supernatant by TCA and then perform my IP in a smaller volume? Or will TCA prevent my antigen from binding to the antibody?
A Even if your protein of interest were still in the supernatant, TCA would precipitate any antibody added to the supernatant. If you do IP, you need to use the conditioned medium from the cell culture.
A TCA is very acidic. It will precipitate any protein regardless of the initial pH of the protein solution. TCA will interfere with the antibody-antigen interaction.
IP can be done in the presence of other proteins, but it should not be done after TCA precipitation. The best way to do it would be to start with about 0.5 mL of your conditioned medium.
A Why not use an anti-BSA antibody to immunoprecipitate the BSA?
A Try to add the SDS-PAGE sample buffer directly to your pellet. Pipet up and down vigorously to break up the pellet. Then heat the solution for 5 min at 95°C and the pellet should go in the solution. That will enable you to get your pellet into a gel for Western blot.
A The amount of BSA in the sample will cause overloading. If you use less sample, the protein of interest will become too low to be detected.
From my perspective, you only have two choices: IP or Blue albumin removal kit. Anti-BSA will be too costly.
A A 5-mL SP column is similar in cost to TCA or acetone protocols.
Q What is the maximum percentage of saturated ammonium sulfate that will result in precipitation of most proteins? Is there a rule to calculate approximately the minimum percentage of saturated ammonium sulfate that will salt out your protein of interest based on its amino acid sequence?
A You might be able to use the Bradford method by salting out prior to dialysis, although I am not sure if the high salt concentration will interfere with the assay.
A Bradford is compatible with up to 1 M ammonium sulfate.
Will you start with the sample including ammonium sulfate or the resuspended, but not yet dialyzed, pellet from ammonium sulfate precipitation?
I recommend the latter. It should work well as long as you have removed all of the super-natant prior to resuspension with fresh buffer.
You might still be able to do the experiment if you have the sample including the ammonium sulfate, but it will depend on the concentration of ammonium sulfate added and how much you dilute it for the assay.
