protein aggregation - prevention/inhibition (Jan/16/2003 )
i have problem in thrombin cleavage, where by my desired protein concentration after cleavage will be very less,
even though there is lot of protein bound to beads, and also cleaved by thrombin
the protein of interest eluted was very less, this may be due to protein aggregation or precipitation,
the protein precipitation is very less/negligible
can any one tell how to prevent/inhibit protein aggregation.
hi
I am also facing the same problem if anybody has any useful information plz let me know.
thanks
sang_tiwari@yahoo.com
hi
maybe this answer will help?...
when i had to resuspend proteinase K, i followed the maniatis protocol. That drived precip of the protein. The tought point was a pH : manufacturer told prot was to be resuspended in acidic solution and then pH should be adjusted.
Mybe pH is case sensitive in your exp?
others ideas
Do you use DTT? check about you reducing agent
These came from a topic i read :
10% glycerol
reduce the possibility of protein aggregation, non specific binding to affinity beads, or protein bining partners, keep your protein's solubility.
0.1% NP-40 as a detergeant
prevent aggregation, hydrophobic interactions between patches
Hi
Thanks for the suggestions i will try these .
maybe this answer will help?...
when i had to resuspend proteinase K, i followed the maniatis protocol. That drived precip of the protein. The tought point was a pH : manufacturer told prot was to be resuspended in acidic solution and then pH should be adjusted.
Mybe pH is case sensitive in your exp?
others ideas
Do you use DTT? check about you reducing agent
These came from a topic i read :
10% glycerol
reduce the possibility of protein aggregation, non specific binding to affinity beads, or protein bining partners, keep your protein's solubility.
0.1% NP-40 as a detergeant
prevent aggregation, hydrophobic interactions between patches
even though there is lot of protein bound to beads, and also cleaved by thrombin
the protein of interest eluted was very less, this may be due to protein aggregation or precipitation,
the protein precipitation is very less/negligible
can any one tell how to prevent/inhibit protein aggregation.
Could u include SDS along.. in the matrix? around 0.1% would also work well..
Do tell us if u could and it worked:)
G'luck!