Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread mesters
Please have a look at the very elaborated/detailed discussion by Martin Chaplin on chaotropes and kosmotropes http://www1.lsbu.ac.uk/water/kosmotropes_chaotropes.html and on the Hofmeister series http://www1.lsbu.ac.uk/water/hofmeister_series.html and *note the diference* in the way the ions

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread Edward A. Berry
On 03/30/2017 08:10 AM, mesters wrote: If the pI of the protein is below the pH of the buffer (net negatively charged protein), optimum stabilization (salting out; lower solubility) of the macromolecule is achieved by combining a kosmotropic anion with a chaotropic cation, e.g.

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread Debanu
gleScholar > Twitter > > Check out my latest paper!!! > The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Akilandeswari > Gopalan [akilaibt2...@gmail.com] > Sent: 30 March 2017 07:02 &g

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread Kittikhun Wangkanont
Hi Akila, I'm curious about your choice of pET22b. If you cut out the signal peptide that comes with the vector and express your protein in the cytoplasm, then what I am about to say doesn't apply. However, if you are exporting the protein into the periplasm, have you considered doing osmotic

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread mesters
If the pI of the protein is below the pH of the buffer (net negatively charged protein), optimum stabilization (salting out; lower solubility) of the macromolecule is achieved by combining a kosmotropic anion with a chaotropic cation, e.g. Ammoniumsulfate (most successful salt)! /For your pI

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread Antonio Ariza
eswari Gopalan [akilaibt2...@gmail.com] Sent: 30 March 2017 07:02 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein precipitation reg Dear all, I have used the following buffers for purification and dialysis. this is fyi. Lysis buffer: 25mM Tris pH 7 or 7.5 or 8 100-500mM NaCl (increase in sal

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread Antonio Ariza
of Chun Luo [c...@accelagen.com] Sent: 29 March 2017 22:15 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg In addition to price, the prevalence of Ni purification may be another reason for Tris popularity. Some His-tagged constructs don't bind to Ni well

[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-30 Thread Hughes, Jon
products -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark Wilson Gesendet: Mittwoch, 29. März 2017 22:12 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg I heartily concur with Craig. Tris can

[ccp4bb] AW: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-30 Thread Hughes, Jon
IL.AC.UK Betreff: Re: [ccp4bb] [ccp4bb] protein precipitation reg Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an old name for sulfuric acid. > On Mar 29, 2017, at 8:40 PM, Keller, Jacob <kell...@janelia.hhmi.org> wrote: > >> And if we are going

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread Paul Miller
Biology Roosevelt Drive Oxford OX3 7BN Original message >Date: Thu, 30 Mar 2017 11:32:05 +0530 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Akilandeswari >Gopalan <akilaibt2...@gmail.com>) >Subject: Re: [ccp4bb] protein precipitation reg &g

Re: [ccp4bb] protein precipitation reg

2017-03-30 Thread Akilandeswari Gopalan
Dear all, I have used the following buffers for purification and dialysis. this is fyi. *Lysis buffer*: 25mM Tris pH 7 or 7.5 or 8 100-500mM NaCl (increase in salt concentration increased precipitation of the protein in the column itself) *5mM Beta mercaptoethanol * *0.5% Triton X 100 * I

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Debanu Das
Hi Akila, In addition to what others have asked about the dialysis buffer, a few more comments that might help to decide next steps because the precipitation (note precipitation and aggregation are related but not synonymous) may be due to several different or related reasons: 1) At what stage

Re: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-29 Thread CRAIG A BINGMAN
Since I’m in full-on cranky old biochemist mode now, I think that vitriol is an old name for sulfuric acid. > On Mar 29, 2017, at 8:40 PM, Keller, Jacob wrote: > >> And if we are going to pour scorn and vitriol on Tris, why not mention its >> large dpKa/dT of 0.03 pH

Re: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-29 Thread Keller, Jacob
>And if we are going to pour scorn and vitriol on Tris, why not mention its >large dpKa/dT of 0.03 pH units/deg ? Hah! That's what many people are doing when they make buffers: pouring vitriol (HCl) on TRIS! I prefer to pour concentrated HEPES, and get two buffers without adding any extra Cl-.

Re: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-29 Thread Janet Newman
] On Behalf Of CRAIG A BINGMAN Sent: Thursday, 30 March 2017 8:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg > On Mar 29, 2017, at 4:15 PM, Chun Luo <c...@accelagen.com> wrote: > > In addition to price, the prevalence of Ni purification may be

Re: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-29 Thread CRAIG A BINGMAN
> On Mar 29, 2017, at 4:15 PM, Chun Luo wrote: > > In addition to price, the prevalence of Ni purification may be another reason > for Tris popularity. Some His-tagged constructs don't bind to Ni well in > HEPES. I wonder if anyone has similar experience or comments. —Chun

Re: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-29 Thread Chun Luo
@JISCMAIL.AC.UK] On Behalf Of Mark Wilson Sent: Wednesday, March 29, 2017 1:12 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [ccp4bb] protein precipitation reg I heartily concur with Craig. Tris can be a dangerous buffer for many reasons, including those listed below. In addition, as a primary

Re: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-29 Thread Mark Wilson
><jon.hug...@bot3.bio.uni-giessen.de> wrote: > >...it's just a wonderful tradition! there's an interesting description of >the history of tris in maniatis >cheers >jon > >Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im > Auftrag von David Briggs >Gesendet: M

Re: [ccp4bb] [ccp4bb] protein precipitation reg

2017-03-29 Thread CRAIG A BINGMAN
CP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von David Briggs Gesendet: Mittwoch, 29. März 2017 17:53 An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Betreff: Re: [ccp4bb] protein precipitation reg It doesn't cost as much as HEPES, iirc. On Wed, 29 Mar 2017, 16:36 Kel

[ccp4bb] AW: [ccp4bb] protein precipitation reg

2017-03-29 Thread Hughes, Jon
? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] On Behalf Of Roger Rowlett Sent: Wednesday, March 29, 2017 11:10 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] protein precipitation reg What are you dialyzi

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Roger Rowlett
half Of *Roger Rowlett *Sent:* Wednesday, March 29, 2017 11:10 AM *To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> *Subject:* Re: [ccp4bb] protein precipitation reg What are you dialyzing against? Your storage solution should typically be buffered away from the pI and

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread David Briggs
* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Roger > Rowlett > *Sent:* Wednesday, March 29, 2017 11:10 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] protein precipitation reg > > > > What are you dialyzing against? Your storage solut

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Bonsor, Daniel
) 706-7457 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Wednesday, March 29, 2017 11:33 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein precipitation reg A bit off topic, but I’ve always wondered how TRIS got so popular what with it’s pKa

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Keller, Jacob
A bit off topic, but I’ve always wondered how TRIS got so popular what with it’s pKa of 8.3—does anyone know? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Wednesday, March 29, 2017 11:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Roger Rowlett
What are you dialyzing against? Your storage solution should typically be buffered away from the pI and contain at least a small amount of kosmotropic salt, e.g. NaCl. Some proteins will require additional stabilizing/solubilizing agents such as glycerol or reducing agents. FYI, Tris-Cl, pH

Re: [ccp4bb] protein precipitation reg

2017-03-29 Thread Keller, Jacob
And what are you dialyzing it against? JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Akilandeswari Gopalan Sent: Wednesday, March 29, 2017 9:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein precipitation reg Dear all, I am a PhD student doing structural

[ccp4bb] protein precipitation reg

2017-03-29 Thread Akilandeswari Gopalan
Dear all, I am a PhD student doing structural studies on a few proteins from Mycobacterium tuberculosis. The gene encoding the proteins I work on are cloned into pet22b with c terminal His tag. the proteins are expressing well. upon purification I am getting good yield of protein but during