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 Thomas University of Wisconsin, Madison Gellman 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 7.5 >> >> 2. 20mM Tris, 500mM NaCl, 1% Triton-X-100, pH 7.5 >> >> 3. 10mM Tris, 1M NaCl >> >> 4. 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 Lab >> >> Institute of Human Virology >> >> University of Maryland, Baltimore >> >> 725 W Lombard Street N370 >> >> Baltimore >> >> Maryland >> >> MD 21201 >> >> Tel: (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, FRSB > Tel: +44 (0) 114 2159552 <+44%20114%20215%209552> > Email: j.r.say...@shef.ac.uk > http://www.sheffield.ac.uk/iicd/profiles/sayers > >
