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Pellet the proteins!? - (Apr/27/2006 )

I've precipitated some supernatant proteins with acetone and then resuspended the pellet in MQ water. After that I did the classical dialysis and my sample went from 250 microliter to 750 microliters. To concentrate, I spin the sample in the centrifuge and poured out the supernatant, resuspending the result pellet in 200 microliter of the supernatant that was left. And i got better amount of proteins than when I just dialyze them and then test on Comassie.

MY PI couldn't believe I've done it, since she says there is no way to concentrate proteins by just spinning them in the centrifuge. Why?

Now I am using Microcon spin columns but I am not getting good results. I cannot concentrate the proteins well enough.

-smoochiepie79-

QUOTE (smoochiepie79 @ Apr 27 2006, 02:21 PM)
I've precipitated some supernatant proteins with acetone and then resuspended the pellet in MQ water. After that I did the classical dialysis and my sample went from 250 microliter to 750 microliters. To concentrate, I spin the sample in the centrifuge and poured out the supernatant, resuspending the result pellet in 200 microliter of the supernatant that was left. And i got better amount of proteins than when I just dialyze them and then test on Comassie.

MY PI couldn't believe I've done it, since she says there is no way to concentrate proteins by just spinning them in the centrifuge. Why?

Now I am using Microcon spin columns but I am not getting good results. I cannot concentrate the proteins well enough.


Sounds like centrifugation with something about 10000g? There is not enought g-force for "pelleting" your protein. A lot of protein was poured away with the supernant. The pellet was only the not solutiable rest of the precipitation with acetone wink.gif

-ms-olli-

Hi!
Acetone acts in the same manner as ethanol and salt precipitation. It will allow the proteins to be pulled down during solvation in centrifuge spin. I sometimes do this at 14K rpm as I do my DNA. Both works fine doing this. I think acetone may be a little harsh, but definately works.

-biokmst-

You can lyophilize the proteins by placing the open microfuge tube in a vaccum lyophilizer. Then reconstitute in the desired amount of the buffer/lysate.

-Rohits-

QUOTE (biokmst @ Apr 27 2006, 07:21 PM)
Hi!
Acetone acts in the same manner as ethanol and salt precipitation. It will allow the proteins to be pulled down during solvation in centrifuge spin. I sometimes do this at 14K rpm as I do my DNA. Both works fine doing this. I think acetone may be a little harsh, but definately works.


Your´re right, but after precip smoochiepie79 resuspend the pellet and dialysis it! There is no precipitated protein for spining down!

-ms-olli-