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problems in concentration protein solution - (May/02/2006 )

hi,

hope that someone can help me or have an idea for solving my problem. i want to concentrate my protein solution by using amicon filter units but unfortunateley most of the protein is in the eluate after centrifugation ohmy.gif and the retentate recovery is low: i've checked 5 µl from the 100µl concentrate versus 10 µl from the 3500µl eluate on sds gel and stained it with coomassie. if i multiplicate the signal from the eluate and compare it to the concentrate it's lousy!
my protein got a his-tag, it is ~ 60 kda and is isolated under denaturated conditions. the elution buffer contains NaH2PO4, Tris-HCL, NaCl, Urea and imidazole. i used a amicon filter unit with 30,000 NMWL following the manufactors instructions.
anyone out there who can help me? sad.gif
thanks in advance for your advices!!
flausch

-flausch-

Maybe get one with even lower NMWL (MWCO)? Three times lower is recommended for >99% recovery.

-K.B.-

QUOTE (K.B. @ May 2 2006, 03:14 PM)
Maybe get one with even lower NMWL (MWCO)? Three times lower is recommended for >99% recovery.



hi k.b.

thanks for your answer! okay, sorry, i forgott to tell, that i have some "contaminating" protein fragments (~ 30kda) in my isolation. but i just want to have the whole protein. so i discussed this point you mentioned with the man from millipore and he decided to use the 30,000 NMWL (=MWCO) with a little chance to eliminate the fragments. and yes, you're wright that three times lower is recommended for 99% recovery and two times lower be said to give a 90% recovery but a look at my gel tells me something else... huh.gif
flausch

-flausch-

Ultrafiltration is OK for concentration but I would not use it for separation of proteins. Maybe concentrate using lower MWCO (10k?) and then separate your protein from contaminating one by gel filtration?

-K.B.-

QUOTE (K.B. @ May 2 2006, 04:58 PM)
Ultrafiltration is OK for concentration but I would not use it for separation of proteins. Maybe concentrate using lower MWCO (10k?) and then separate your protein from contaminating one by gel filtration?



hi k.b.
but to my information i need a protein eluter for this step or do you have a protocol for excising the band out of the sds gel and resolving the protein?
flausch

-flausch-

this is probably a dumb question but I need to make sure...you're not eluting your protein off the amicon column, are you?

-aimikins-

QUOTE (flausch @ May 2 2006, 05:22 PM)
but to my information i need a protein eluter for this step or do you have a protocol for excising the band out of the sds gel and resolving the protein?

I don't understand your question. Haven't you mistaken gel filtration (eg. Sephadex) with gel electrophoresis?

-K.B.-

QUOTE (K.B. @ May 3 2006, 01:05 PM)
QUOTE (flausch @ May 2 2006, 05:22 PM)

but to my information i need a protein eluter for this step or do you have a protocol for excising the band out of the sds gel and resolving the protein?

I don't understand your question. Haven't you mistaken gel filtration (eg. Sephadex) with gel electrophoresis?



hi k.b.

sorry, my fault blink.gif .
unfortunately i have no experience with gel filtration like sephadex. i have looked at the amersham catalogue for information about this application but it was not really helpfull. can you explain it in a few sentences or give me an advice where i can get more information eg. the requirements for this or how to work with this?
sorry but we're new on this exciting field "proteins expression, isolation and purification" and i have to it by my own just with information from books or the world-wide-web. so any advices will be highly appreciated smile.gif thanks!!

-flausch-

QUOTE (aimikins @ May 2 2006, 09:35 PM)
this is probably a dumb question but I need to make sure...you're not eluting your protein off the amicon column, are you?



hi aimikins,

sorry, but i don't understand your question huh.gif

-flausch-