We are currently working with Jurkat cells, when performing western blot analysis the bands come out quite weak... the protein concentration we have been able to obtain from whole cell lysate is always relatively low (0.93-1,79 mg/ml) compared to LCL cells which we also work with (always >2mg/ml)
The protocol we're using for preparing WCL calls for 1ml Ripa for 2x10^7 cells, and a ratio of 1:7 Protease inhibitor to Ripa.
Is there any way in which we could improve the resultant protein concentration?
Thank you :)
Dear Dr. Belén Garcia Fariña
You may try out the following suggestions.
1. Try to reduce the volume of RIPA lysis buffer to obtain higher protein concentration. I feel 1ml of RIPA is too high. Incubate the cells in RIPA on ice for 30 min, vortexing occasionally. Ensure that the pellet is fully suspended in RIPA. If needed, extend the incubation time.
2. Instead of a protease inhibitor, add the protease inhibitor cocktail which is used for the instantaneous protection of proteins from various proteases during cell extraction procedure. It is supplied as a 100X solution. The dilution of the cocktail that is to be used will be provided by the manufacturer, which is usually based on the volume of the sample. Add protease inhibitors fresh to the lysis buffer just before carrying out cell lysis.
3. Alternatively, you may prepare stock solutions of protease inhibitors in-house, if you have the necessary chemicals, as follows.
Protease Inhibitors:
Aprotinin: Dissolve 20 mg in 10 ml of water or PBS to get 2mg/ml stock (1000X). Working concentration is 2µg/ml, so add 1µl of stock in 1 ml of lysis buffer.
Leupeptin: Dissolve 50 mg in 10.5 ml of water to get 10 mM stock (1000X). Working concentration is 10 µM, so add 1µl of stock in 1 ml of lysis buffer.
Pepstatin: Dissolve 5 mg in 7.3 ml of Methanol or DMSO to get 1 mM stock (1000X). Working concentration is 1µM, so add 1µl of stock in 1 ml of lysis buffer.
PMSF: Add 17.4 mg of PMSF per ml of isopropanol to prepare 100 mM solution. Store at −20°C. PMSF is inactivated in aqueous solutions. Use 0.1-1mM in final volume of your lysis buffer.
Aprotinin is a broad-spectrum serine protease inhibitor.
Leupeptin is an inhibitor of various proteases. It will inhibit serine, cysteine, and threonine proteases.
Pepstatin is a highly selective inhibitor of acid proteases (aspartate proteases).
PMSF is a serine protease inhibitor. PMSF does not inactivate all serine proteases.
Try adding all the above inhibitors to the ice-cold RIPA lysis buffer, fresh, just before you carry out cell lysis. It will help!
Regards,
Malcolm Nobre
Thank you very much for the thorough explanation! Next time I do WCL I'll try to do it this way :)
Best regards,
Belén