getting negative protein measurements - (Nov/21/2012 )
I've been having some trouble with my cell lysates in preparation for a western.
I collected my cells using RIPA buffer but after spinning them down to get rid of the nucleus, no pellet seems to show up.
We've had problems with our RIPA buffer before and finally got it fixed and this is the first time for me to use it ever since.
I went ahead and did the protein measurements using Bio-Rad assay but as expected, the measurements turned out to be very low or negative.
This is the second time in a row this has happened and I have no idea why the cells aren't getting lysed.
Any suggestions or help?
Check that the buffer components are compatible with bradford assay (assuming you are using the biorad bradford), this is especially important for the concentrations of detergents.
Make sure that the kit hasn't expired.
Assuming you did biorad bradford:
deejay on Wed Nov 21 18:50:04 2012 said:
I collected my cells using RIPA buffer
what do you mean you collected them with RIPA?
to add to bob1's comment I think your problem is lysis, not Bradford. First you must trypsinize or scrape your cells, centrifuge to pellet them down, then add 'cold' RIPA which has protease inhibitor. rotate on a rotator at 4C for a while (10 min enough, or you can just keep on ice but don't pipet up and down, it might give you a gooey DNA clump) and then centrifuge at high speed for 5 min. I must add that RIPA is a strong lysis buffer and breaks down all cell membrane including nuclei membrane. So the pellet at this stage is cellular debris including DNA, not intact nuclei. If you have collected a lot of cells then you must see a big pellet, if you didn't have a lot of cells then the pellet is smaller.
Also how much RIPA are you adding? for a T25 flask 100-1000 ul is enough.
I did Bradford on microplate, and I only got negative readings in my empty wells. my first standard sample was set zero.