Thanks to everyone who responded to my post (especially, John Lee and Brad
Bennett) with helpful comments and suggestions. The good news is that I was
able to get the Ulp1 protease cleavage step to 100% completion at 4C by
using between 1:3-to-1:2 enzyme:substrate ratios and incubating the
reaction
Dear Raji,
In our lab we always
(1) use TCEP
(2) high molar ratio of protease with gentle rocking at 4C O/N
(3) keep in mind the size (KDa) of the target protein regarding to the
detergent micelle size. Although has been observed that in many cases the use
of SUMO tags increases expression
Hi Raji-
To echo what John Lee said, we found that we had to maintain a ratio
between 1:2 and 1:10 Ulp1:substrate for several different target proteins,
none of which were membrane proteins however. So, I would definitely try
maybe a 1:5 ratio and see what happens. This wasn't too cost prohibitive
Hi Folks,
Thanks very much for your helpful suggestions. Several folks have suggested
switching to TCEP. I am also going to increase the molar ratio of Ulp1 in
the cleavage reaction like John Lee has suggested since I did not think of
the effect of DDM on Ulp1. (Everything seemed to work in the ca
Hi Raji,
I've used SUMO/Ulp1 for some of my membrane proteins. My experience is you
have to add a lot of protease. Sometimes, I add 1-1 molar ratio to get the
job done.
Just to add, I've experienced similar results as you in 0.1%DDM, partly
because Ulp1 precipitates in the detergent (worse in OG),
Sorry for my typo, it is Ulp1-SMT3 complex...
On Sun, Feb 16, 2014 at 10:54 PM, Chen Zhao wrote:
> By the way, I have an unrelated question. In the crystal structures
> containing yeast SUMO (including Ulp1-SMT1 complex: 1EUV), the first ~19
> residues are absent. I am curious whether people tr
By the way, I have an unrelated question. In the crystal structures
containing yeast SUMO (including Ulp1-SMT1 complex: 1EUV), the first ~19
residues are absent. I am curious whether people tried a SUMO tag with
these residues deleted. I am using the vector from invitrogen which seems
to have the f
You could also try TCEP as a reducing agent-strong and compatible with IMAC.
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji
Edayathumangalam
Sent: Sunday, February 16, 2014 10:58 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Trouble cleaving SUMO tag off of
Hi Raji,
I have no experience with membrane proteins, but I have used SUMO tags
frequently. Unlike other proteases that cleave at a specific site
(thrombin, TEV, etc.), Ulp1 recognizes the tertiary structure of SUMO for
cleavage. So if only about 50% of your protein is cleaved, this may
indicate
Hi Raji,
Your membrane protein should be in micelles as a protein-detergent
complex. The detergent belt buries much of the protein surface area and can
make a lot of biochemistry experiments less efficient.
I've never worked with SUMO tags before, but if cleaving them is like
with oth
Hi Everyone,
After several attempts to cleave the SUMO tag off my membrane protein under
various conditions (different reducing agents, enzyme-to-substrate ratios,
etc.) and after reading the manual and troubleshooting guide, I'm reaching
out to the ccp4bb community.
Experiment: I dialyze the mix
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