Re: [ccp4bb] Problems with an exonuclease

2017-06-08 Thread Mohammad Khan
Dear all, I apologize that till now I was making the mistake of not sending the replies in the common thread, but somehow only to each person individually. I am summarizing my answers since yesterday here: I have tried refolding it at pH 7, 7.5 and 8. I add 4mM TCEP to my urea buffers and reduce

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread John Newitt
Probably nucleic acids. Increase the number or volume of washes and improve the washing of your inclusion bodies. Instead of sonication, we use a Polytron homogenizer to resuspend the IBs pellet during washing. This is faster and easier. Incorporate an additional chromatography step such as Heparin

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread Edward A. Berry
On 06/07/2017 10:46 AM, Bonsor, Daniel wrote: It will either be two things. DNA or residual Triton-X-100. Actually Triton X-100 absorbs more at 280 than 260 - in fact the hydroxyphenyl in TX-100 looks remarkably like tyrosine in RNase A. I long ago gave up hope of resolving triton from protei

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread Bonsor, Daniel
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

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread S. Mohanty
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 I

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread Nicole Thomas
;> >> Institute of Human Virology >> >> University of Maryland, Baltimore >> >> 725 W Lombard Street N370 >> >> Baltimore >> >> Maryland >> >> MD 21201 >> >> Tel: (410) 706-7457 >> >> >> >> >&

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread Jon R Sayers
t; 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 > > > >

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread Bonsor, Daniel
@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

Re: [ccp4bb] Problems with an exonuclease

2017-06-07 Thread Jon R Sayers
Dear Mohammad, If your protein is purified from insoluble material there could be some DNA in there though if it were stoichiometric your 26 would be >> than your 280, as the former has a much higher extinction co-efficient. A ratio of 2 is could be RNA contamination. I'd also check the mass spec

[ccp4bb] Problems with an exonuclease

2017-06-07 Thread Mohammad Khan
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 aggrega