I have performed several western blots using lysates from umbilical cord white blood cells that have been treated for about 48hrs and lysed with RIPA buffer (Halt protease inhbitor, 10mM PMSF and 10mM Na3VO4). I usually start with 1-5 million cells and lyse them in 60-100uL of this RIPA buffer + protease inhibitors. My protein of interest is the glucocorticoid receptor (GR). I have been seeing multiple bands that are smaller than the main GR isoform. I tried different antibodies and got similar results. I have also been seeing non-specific bands for other proteins as well. I initially thought they were isoforms but after trying another lysis method (8M Urea), which should completely denature all proteins, I do not see those other bands anymore. I am thinking that perhaps there are some proteases that are not sufficiently inhibited and so are degrading my protein. Has anyone experienced something like this or can you recommend a protease inhibitor cocktail that has worked well for PBMCs and/or white blood cells?
Hello Nana Abena Anti
Yes, you may be right. There may be some proteases that are not sufficiently inhibited and may be degrading your protein. Cells contain many different types of proteases. When you lyse cells either by adding detergents or by physically disrupting the cells, not only do you release your protein, but you also release a variety of other proteins that can be harmful to your protein. So, you need to add a mixture of protease inhibitors to your lysis buffer. Therefore, mixture of different protease inhibitors is needed for complete protection of proteins during isolation for subsequent experiments like western blotting.
However, since there is not one chemical that can effectively deactivate each known protease, most of the researchers prefer to use ready-to-use protease inhibitor cocktail. Instead of using the protease inhibitor cocktail, you could prepare your own mixture of several inhibitor compounds in the laboratory.
PMSF will work by deactivating the serine hydroxyl group and any other enzyme that contains serine in its active site through an esterification process. You should try adding other protease inhibitors like aprotinin, leupeptin, EDTA in addition to PMSF which would help. Add the inhibitors to the lysis buffer fresh just before you begin cell lysis. Always carry out the cell lysis on ice as the activity of proteases is reduced at 4°C.
You may use other protease inhibitors as given below:
Aprotinin: It is a bovine pancreatic trypsin inhibitor that inhibits serine proteases, like trypsin, chymotrypsin and plasmin. Aprotinin is a reversible inhibitor. Make stock solution of aprotinin in water (5 mg/mL) and store at 4°C for up to one week. A good working concentration is 1-2 µg/mL.
Leupeptin: It reversibly inhibits cysteine, serine and threonine proteases, such as trypsin, plasmin, papain, cathepsin B, and kallikrein. However, it does not inhibit pepsin, cathepsins A and D, elastase, renin, or chymotrypsin. Leupeptin is soluble in water, ethanol, and acetic acid. Store stock solutions (50 mg/mL) at -20°C and use at 1-2 µg/mL.
Pepstatin A: It inhibits aspartyl proteases, such as pepsin, and cathepsins D and E. It is insoluble in water, but stock solution can be made in a variety of alcohols and acetic acid (e.g., 1 mg/mL in methanol) and stored at -20°C. Working concentration (1 µM) is stable for one day at room temperature.
PMSF: It is an irreversible inhibitor of several serine proteases, namely trypsin, chymotrypsin, and thrombin. It can also inhibit some cysteine proteases, metalloproteases, and aspartic proteases. Make the stock solution in an anhydrous liquid such as ethanol (200 mM), store at 4°C and add to your lysis buffer just before use (final concentration 0.1 - 1mM).
EDTA: Metalloproteases are inactivated by EDTA. Use 1-10 mM solutions of EDTA to inhibit proteases.
Sodium orthovanadate (Na3VO4) is a phosphatase inhibitor (Tyr phosphatase and alkaline phosphatase inhibitor), and should be used in the lysis buffer if you are detecting phosphoprotein by Western Blot. You may also add Sodium fluoride which is a ser/thr phosphatase and acid phosphatase inhibitor at a working concentration of 10mM.
Best.
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