Cytochrome C detection by Western Blot - a typical question (Aug/12/2008 )
I need to detect cytochrome c release before and after apoptosis.
Do I need to follow up any specific protocol for mitochondria isolation? or just an ordinary RIPA isolation is enough?
Frankly speaking, I need a protocol for cytoplasmic extraction.
thanks
-Curtis-
LOL, several questions there!
You can't use RIPA because you'll solubilize mitochondria and release all cyt c. You can use a non-detergent hypotonic buffer to swell the cells (eg Tris, no salt or small amount, protease inhibitors), disrupt cells with dounce homogeniser or needle. View the disrupted cells under the scope - should be able to see nucleus and broken bits of cytosol. Spin at low speed eg 3,000 g to pellet the nucleus/mito/unbroken cells. Collect the supernatant.
-Michelle4-
QUOTE (Michelle4 @ Aug 12 2008, 10:34 PM)
LOL, several questions there!
You can't use RIPA because you'll solubilize mitochondria and release all cyt c. You can use a non-detergent hypotonic buffer to swell the cells (eg Tris, no salt or small amount, protease inhibitors), disrupt cells with dounce homogeniser or needle. View the disrupted cells under the scope - should be able to see nucleus and broken bits of cytosol. Spin at low speed eg 3,000 g to pellet the nucleus/mito/unbroken cells. Collect the supernatant.
You can't use RIPA because you'll solubilize mitochondria and release all cyt c. You can use a non-detergent hypotonic buffer to swell the cells (eg Tris, no salt or small amount, protease inhibitors), disrupt cells with dounce homogeniser or needle. View the disrupted cells under the scope - should be able to see nucleus and broken bits of cytosol. Spin at low speed eg 3,000 g to pellet the nucleus/mito/unbroken cells. Collect the supernatant.
Thanks,
just as I thought........RIPA, NP40, Triton X-100 and CHAPS buffers are for total protein extractions.
I don't have Dounce Homogenizer. do you think I can use a Digitonin buffer?
Digitonin buffer is:
190ug/ ml Digitonin
75mM NaCl
1mM NaH2PO4
8mM Na2HPO4
250mM Sucrose
somebody gave this recipe to me and said it'll extract only cytosol.
-Curtis-