Where did the protein go? - Low protein reading in WB lysates (Oct/10/2005 )

I really need help on this one....

I sonicated about 3 million cells ( a good sized pellet) in 100 ul of cyclin ripa buffer with protease inhibitors etc. After I sonicated for 9-10 pulses (as I ussually do) I spun the sample at 12,000 RPM for 15 minutes. When I read my prteon cocncentration using the Bradford assay I got a reading of 2 -3 ug protein/4 ul of the sample.

Using the same procedure described above normaly I get a reading of 4-5 ug/2 ul of the samples. The only thing different that I did today was I spun the sample for 15 mins instead of 10. Will this make a difference. Everything else was the same.

The spectrophotometer was fine as another labmate got her usual results.
Any pointers?? I got so disgusted that I just left from lab...

Anyone??

When you spin longer, you'll remove a bigger amount of cell debris like small pieces of membrane...

This can explain why your protein contect in the soluble fraction was lower... BUT, this is the way to do it not to be mislead by the protein amount.

" BUT, this is the way to do it not to be mislead by the protein amount"

Can u clarify that statement please...Do you mean I have to spin so long??

'Coz ....my lab mate who gets very high protein readings (although she uses half the pellet size and the same amount of the ripa buffer) spins at 6000 RPM for 6-8 mins. Is this the right thing to do??