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
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
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
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
IL.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 solub
Daniel A Bonsor PhD.
>>
>> Sundberg Lab
>>
>> Institute of Human Virology
>>
>> University of Maryland, Baltimore
>>
>> 725 W Lombard Street N370
>>
>> Baltimore
>>
>> Maryland
>>
>> MD 21201
>>
>> Tel
l: (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
>
>
>
> De
@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
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
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
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