Yes, washing IB with 10 % BPER twice with sonication makes our IB (from
various proteins) very clean before denaturation with 6M guanidine
hydrochloride and further processing. Smita
On Wednesday, June 7, 2017 12:03 PM, Nicole Thomas <nctho...@wisc.edu>
wrote:
I've found that washing my IB's with B-PER helps dramatically to get rid of
any impurities.
https://www.thermofisher.com/order/catalog/product/78248
Nicole ThomasUniversity of Wisconsin, MadisonGellman Group
On Wed, Jun 7, 2017 at 11:27 AM, Jon R Sayers <j.r.say...@sheffield.ac.uk>
wrote:
I missed the Triton - that will be it!
On 7 June 2017 at 15:46, Bonsor, Daniel <dbon...@som.umaryland.edu> wrote:
It will either be two things. DNA or residual Triton-X-100. When you say,
cleaned the IBs, do you mean you sonicated the IBs, or just resuspended the
pellet and then centrifuged again? If the latter, try sonication. I wash my IBs
at least 4 times with the following buffers; 1. 20mM Tris, 500mM NaCl, 1%
Triton-X-100, pH 7.52. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.53. 10mM
Tris, 1M NaCl4. 20mM Tris, 500mM NaCl, pH 7.5 By resuspension and then
sonication. This I find removes DNA and Triton-X-100. Also, if the pellet is
very large, you may need to increase the number of washes, volume and length of
sonication or split the pellet up. Other things to try…1. Change the wash
salt to KCl and use more, (3M). I was informed that KCl is a better disrupter
of DNA than NaCl (I stand to be corrected if this is wrong).2. At each
wash stage, dissolve a small amount of IBs and measure the 260/280. The ratio
should decrease in the latter washes, if they are working.3. Does your
exonuclease typically contain a divalent metal? You could try adding EDTA to
the wash steps which may help in preventing DNA stick to your protein. All the
best! Dan Daniel A Bonsor PhD.Sundberg LabInstitute of Human
VirologyUniversity of Maryland, Baltimore725 W Lombard Street
N370BaltimoreMarylandMD 21201Tel: (410) 706-7457 From: CCP4 bulletin board
[mailto:CCP4BB@JISCMAIL.AC.UK]On Behalf Of Mohammad Khan
Sent: Wednesday, June 07, 2017 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with an exonuclease Dear all, I am working with an
exonuclease by refolding it from inclusion bodies (IBs). I tried various
constructs and hosts, but couldn't get it in soluble form. I lyse my cells
using a cell disruptor and after solubilizing IBs with urea, I refold the
protein by rapid dilution and get an aggregate and monomer peak of the same on
GFC. and have checked CD as well as activity, both of which are good. My issues
is as follows: I get a high 260 nm peak while purifying it on GFC. the 260/280
ratio can reach upto 2. I have tried all means to get rid of watever this
contamination is: cleaned the IBs with 1% Triton X-100, 2 M NaCl, added Dnase
prior to lysis. I have also used methods to remove the DNA from protein, if
that is the contaminating agent. I am trying to crystallize the protein with no
success so far.Moreover, my thermofluor assays give very low fluorescence. I
use Sypro Orange as a fluorophore. Suprisingly, a point mutation in the active
site (His to Arg) gets rid of the issue of contamination and gives me good
thermofluor curves. I purify the mutant also form IBs. Can someone suggest
what this "contamination" may be? Thank you for your time.
--
Best wishes
Prof. Jon R Sayers, FRSBTel: +44 (0) 114 2159552
Email: j.r.say...@shef.ac.ukhttp://www.sheffield.ac.uk/ iicd/profiles/sayers
