Dear Dr. Amira A. Abdellatef,
Yes, you may use RIPA buffer. But the choice of lysis buffer to be used should be such that it ensures that the protein of interest is stable, soluble, and not aggregated.
RIPA buffer will lyse your cells releasing all proteins within compartments, including nuclear and mitochondrial proteins. This is due to the combination of harsh denaturing, ionic detergents (sodium deoxycholate and SDS) and the milder, non-ionic detergent (NP-40).
When you use RIPA buffer, you will solubilize nuclear membrane releasing genomic DNA. This in turn will make your sample into a gooey mess and it will be very difficult for you to pipette.
NP-40 and Triton X-100 are milder, non-ionic detergents. They are good at solubilizing membrane proteins and for isolating cytoplasmic proteins. Proteins retain their native state in the presence of these detergents and protein-protein interactions can be preserved. If you are interested in isolating the nuclear fraction, you may do so by centrifuglng the lysate and resuspending the nuclear pellet in RIPA buffer.
If you wish to extract only the cytoplasmic proteins, you may incubate the cells in the following ice cold lysis buffer. The recipe is given below.
25mM Tris pH 7.5,
100mM NaCl,
5mM EDTA,
0.5% NP-40, and
1mM PMSF (to be added fresh at the time of cell lysis)
for 10 - 15 mins, and then centrifuge the cell lysate at 15000 x g for 15 mins at 4 degree C.
The volume of lysis buffer should be adjusted depending on the volume of insect cells used for a particular experimental condition. For example, 1ml of lysis buffer should be used for 2 x 10^7 cells from insect culture.
Regards,
Malcolm Nobre
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