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Pellet after centrifuging - (Apr/27/2006 )

Hello, I have a question. I am doing work on rat striatum and we homogenize them in sample buffer. 1 ml of (50mM tris-HCl ph 7.0, 2% SDS, 50 mM DTT) sample buffer per 40-60 mg of tissue. To this buffer I add 10 ul Phosphatase inhibitor cocktail 1 and 2 from sigma per 1 ml of buffer and 5 ul of Protease inhibitor from sigma per 1 ml of buffer.
We sonicate the pellet for 5 to 15 seconds until homogenized. Then we boil the sample for 10 min then cool on ice for 5 min and then centrifuge at 14K RPM for 15 min at room temp. The supernatant is removed and then spun again for 15 min at 14K RPM.
The problem is that after freezing and then later when I go to use it if I spin it again I get more precipitate and I am assuming this might be causing the unevenness in my control bands.
So I suppose my question is 1. Do I need to spin at a higher RPM or longer to get rid of all the soluble matter but still retain my proteins (ERK 1/2 42/44 kDa, tyrosine hydroxylase, c-Fos, Creb, actin, and D2 receptors 90 kDa)? 2. Do you think this extra soluble matter could cause non uniform bands?

-Phalen-

could it be the SDS that precipitates?
treat the buffer the same way than the sample and you will see.

-Missele-

QUOTE (Missele @ Apr 28 2006, 03:09 AM)
could it be the SDS that precipitates?
treat the buffer the same way than the sample and you will see.

Nope, one of the other people in my lab tried that and there was no precipitate in just the sample buffer.

-Phalen-