User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13: Difference between revisions
No edit summary |
|||
| Line 10: | Line 10: | ||
The Pierce Refolding Kit Instructions<cite>Pierce</cite> that detail the 9 buffer components in their kit, which are: | The Pierce Refolding Kit Instructions<cite>Pierce</cite> that detail the 9 buffer components in their kit, which are: | ||
reducing agents | reducing agents<BR> | ||
PEG | PEG<BR> | ||
divalent cations | divalent cations<BR> | ||
high [NaCl] | high [NaCl]<BR> | ||
L-arginine | L-arginine<BR> | ||
guanidinium chloride (GnCl) | guanidinium chloride (GnCl)<BR> | ||
<BR> | |||
and an unreleased '''"Refolding Guide"''' that comes with the ProMatrix kit an is not available electronically | and an unreleased '''"Refolding Guide"''' that comes with the ProMatrix kit an is not available electronically | ||
<BR><BR> | |||
----<BR> | |||
A paper on High throughput automated refolding <cite>Vincentelli</cite> that describes a 96 well matrix of buffer conditions they developed for structural genomics projects for Mtb and Viruses. <BR> | |||
Finally, Vertex Pharmaceuticals <cite>Willis</cite> fills the gaps and describes the use statistical software to generate a fractional factorial screen; application of light absorption to measure protein precipitation (λ 390nm); and most '''importantly''' statistical analysis of the data (both precipitation and enzyme activity)from the fractional factorial matrix to draw conclusions and further direct optimization.<BR> | |||
<BR><BR><BR> | |||
---- | ---- | ||
<BR> | |||
==Designing Our Matrix== | ==Designing Our Matrix== | ||
<BR> | |||
The problem with any matrix report, is that they are packed full of misc. chemicals that are either not in house, or prohibitively expensive... it is my experience to find a way to furnish results, then ask for $$$ from the boss... | The problem with any matrix report, is that they are packed full of misc. chemicals that are either not in house, or prohibitively expensive... it is my experience to find a way to furnish results, then ask for $$$ from the boss... | ||
<BR> | |||
This excludes: | This excludes: | ||
all Non Detergent SulfoBetaines (NDSB's) that can or cannot be important | <BR>all Non Detergent SulfoBetaines (NDSB's) that can or cannot be important | ||
a-cyclodextrin and methyl-B-cyclodextrin (NB: B-cyclodextrin that we have in house is not soluble in H<sub>2</sub>O.) | <BR>a-cyclodextrin and methyl-B-cyclodextrin (NB: B-cyclodextrin that we have in house is not soluble in H<sub>2</sub>O.) | ||
fancy detergents (lauryl maltoside) | <BR>fancy detergents (lauryl maltoside) | ||
N-lauryl sarcosine (iFOLD kits) | <BR>N-lauryl sarcosine (iFOLD kits) | ||
FoldACEs (iFOLD series 3 kit) | <BR>FoldACEs (iFOLD series 3 kit) | ||
disco reducing agents (BMC: ''bis''-mercaptoacetamide cyclohexane) | <BR>disco reducing agents (BMC: ''bis''-mercaptoacetamide cyclohexane) | ||
<BR><BR> | |||
additionally, | additionally, <BR> | ||
<BR> | |||
From Willis' <cite>Willis</cite> report, buffer pH, detergent, and NDSB 201 were important for some proteins,<BR><BR> | |||
So, our matrix will include an expansion of pH, and various detergents around that may or may not help (it's a screen for a reason) | |||
additives that are not NDSB's that may help, and some other stuff I like to hypothesize about.<BR> | |||
<BR> | |||
The conditions we will screen follow:<BR> | |||
<BR> | |||
<u>pH/ Buffer</u><BR> | |||
==References== | ==References== | ||
Revision as of 11:30, 13 October 2009
 Refolding Matrix
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
In the beginning there was Jackgetting started here... The background reading I have done that will weigh heavy today: The Pierce Refolding Kit Instructions[1] that detail the 9 buffer components in their kit, which are: reducing agents A paper on High throughput automated refolding [2] that describes a 96 well matrix of buffer conditions they developed for structural genomics projects for Mtb and Viruses.
Designing Our Matrix
additionally, So, our matrix will include an expansion of pH, and various detergents around that may or may not help (it's a screen for a reason)
additives that are not NDSB's that may help, and some other stuff I like to hypothesize about. References
| |
