centricon concentrators

Richard Kerr kerrr at CRYPTIC.RCH.UNIMELB.EDU.AU
Wed Sep 3 19:19:22 EST 1997


At 15:10 3/09/97 +0200, you wrote:
>
>Patricia Werner (werner at inw.agrl.ethz.ch) wrote:
>> I hope somebody can help me! I am isolating a protein with His-Tag.
>> In order to get rid of the poly-histidine I am doing a rTEV-protease
>> digestion. The problem is, that in the end of the isolation the protein
>> is in 4M Urea, 500 mM Imidazol, but the protein doesn't work with such 
>> high Urea concentrations.
>> 
>> So I am trying to change the buffer by centrifugation with centricon 
>> concentrators from amicon. But yield is only about 10%. The protein seems
>> to dissapear during the concentration procedure. I can find it neither 
>> in the flow-through, nor in the retentate. 
>
>Sounds like the protein is precipitating on the membrane. Maybe you could
>try dialysis instead.

The protein doesn't have to precipitate on the membrane (merely 'stick') to
decrease the yield. in order to make the membrane less sticky, you have a
few alternatives.

1. flush about 1 volume of your buffer plus 0.01% triton x 100 or 0.5% CHAPS
thru' the membrane before you use it...the detergents help condition the
membrane.

2. flush thru' some carrier protein to block the stickiness of the membrane...
eg if your membrane cut off is 100 kD, use BSA, if it is 30 kD use something
like haemoglobin

3. it sounds like you want to recover the protein for an activity assay
rather than to check its purity......why not put some carrier protein (BSA
or OVALBUMEN) in with the protein as it is being concentrated??

dialysis is an option, but its very slow and you have no garuntee that your
protein will stay in solution of active after you dialyse.



>Can't you exchange the buffer on the column? I.e. eluting in Imidazol
>without the urea?

This approach has the same draw backs as the centricon approach, with the
added negatives that it is slow , you have to concentrate the sample agian
at the end of the procedure and when you buffer exchange the protein may
stick to the beads of the column.
The other alternative is that your protein may be really 'sticky' due to its
primary structure.  What does your protein look like? does it have any runs
of hydrophobic residues?
have you done a hydrophobicity plot to check ?
What purpose is the urea serving ?



>/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
>/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP3 */
>/* "Science is the game we play with God to find out what His rules are."  */

As a small flame-bait; who is this God person ? ;->
Richard Kerr.
The Murdoch Institute,
R.C.H. Flemington Rd, Parkville, 3052,
AUSTRALIA.
kerrr at cryptic.rch.unimelb.edu.au
Phone (61) 3 9345 5045.
FAX   (61) 3 9348 1391.
'The most interesting things about vertebrates occur in the neural crest.'
	Peter Thorogood.




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