User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13: Difference between revisions

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guanidinium chloride (GnCl)<BR>
guanidinium chloride (GnCl)<BR>
<BR>
<BR>
and an unreleased '''"Refolding Guide"''' that comes with the ProMatrix kit an is not available electronically
also contained in the kit is an unreleased '''"Refolding Guide"''' that comes with the ProMatrix kit an is not available electronically... it would be nice to read this, but C'est la vie!
<BR><BR>
<BR><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>
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>
----
<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>
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><BR><BR>
Line 84: Line 86:
<BR>
<BR>
<BR>
<BR>
Why BSA? it just works for me.<BR>
Why BSA? this is my screen, and a I want to try something wacky.<BR>
===Stock Solutions===
===Stock Solutions===
it is important to make sure that your stocks are at concentrations appropriate such that an all positive sample (e.g. one having all possible components) does not have a volume greater than your total volume for the experiment
it is important to make sure that your stocks are at concentrations appropriate such that an all positive sample (e.g. one having all possible components) does not have a volume greater than your total volume for the experiment
 
<BR><BR>
here is the table I cooked up to figure out what "X" concentration I would need to make everything work, taking into account certain characteristics (e.g. if 20% glycerol will be the final concentration, then i cannot achieve more than a 5X stock solution)
here is the table I cooked up to figure out what "X" concentration I would need to make everything work, taking into account certain characteristics (e.g. if 20% glycerol will be the final concentration, then i cannot achieve more than a 5X stock solution)
{| {{table}}
<BR><BR>{| {{table}}
| align="center" style="background:#f0f0f0;"|'''component'''
| align="center" style="background:#f0f0f0;"|'''component'''
| align="center" style="background:#f0f0f0;"|'''stock solution'''
| align="center" style="background:#f0f0f0;"|'''stock solution'''
Line 121: Line 123:
|}
|}
''NB: I used the totally wicked [http://excel2wiki.net/index.php excel2wiki] converter for the table code''
''NB: I used the totally wicked [http://excel2wiki.net/index.php excel2wiki] converter for the table code''
<BR><BR>
so we are set.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''component'''
| align="center" style="background:#f0f0f0;"|'''concentrations from Willis'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
| Buffer||50||mM||
|-
| detergent||0.5||mM||~0.06%
|-
| amino acids||550||mM||
|-
| GnCl||550||mM||
|-
| Ligand||100||uM||
|-
| PEG||0.06||%||
|-
| Glycerol||20||%||
|-
| Salt MIX||264/11||mM Na/K||
|-
| BSA||10||mg/mL||
|-
| Reducing agent||5||mM||
|-
|
|}
stock solutions we will need:




Revision as of 12:42, 13 October 2009

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In the beginning there was Jack

getting 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
PEG
divalent cations
high [NaCl]
L-arginine
guanidinium chloride (GnCl)

also contained in the kit is an unreleased "Refolding Guide" that comes with the ProMatrix kit an is not available electronically... it would be nice to read this, but C'est la vie!


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.


Finally, Vertex Pharmaceuticals [3] 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.




Designing Our Matrix


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 that are desired first, then ask for $$$ chemicals to improve future experiments second...
This excludes:


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 H2O.)
fancy detergents (lauryl maltoside)
N-lauryl sarcosine (iFOLD kits)
FoldACEs (iFOLD series 3 kit)
disco reducing agents (BMC: bis-mercaptoacetamide cyclohexane)

additionally,

From Willis' [3] report, buffer pH, detergent, and NDSB 201 were important for some proteins,

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.

The conditions we will screen follow:

pH/ Buffer
5.5 MES
6.5 MES
7.5 HEPES
8.2 TRIS
9.0 CHES

I replaced BORATE pH 9.5 from [3] with CHES to alleviate complications that will arise during reagent preparation, since borate is only marginally soluble in water, and cannot be prepared as a 10X stock (500 mM).


detergents/ concentration
triton X 100
tween 80
Chaps
none

NB: NONE is a category for all subsequent categories, because it must be included for matrix generation

reducing agents/ concentration
DTT
TCEP
β-ME
none

additives:

arginine
asparate
divalent cations (2 Mg++/ 0.1 Ca++)
GnCl
Ligand
PEG 3350
Glycerol
Sucrose
BSA
NaCl/KCl (high ionic strength)


Why BSA? this is my screen, and a I want to try something wacky.

Stock Solutions

it is important to make sure that your stocks are at concentrations appropriate such that an all positive sample (e.g. one having all possible components) does not have a volume greater than your total volume for the experiment

here is the table I cooked up to figure out what "X" concentration I would need to make everything work, taking into account certain characteristics (e.g. if 20% glycerol will be the final concentration, then i cannot achieve more than a 5X stock solution)

{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;"

component stock solution relative volume
Buffer 10X 10
detergent 100X 1
amino acids 5X 20
GnCl 10X 10
Ligand 10X 10
PEG 100X 1
Glycerol 5X 20
Salt MIX 10X 10
BSA 10X 10
Reducing agent 100X 1
total 93

NB: I used the totally wicked excel2wiki converter for the table code

so we are set.

component concentrations from Willis ' '
Buffer 50 mM
detergent 0.5 mM ~0.06%
amino acids 550 mM
GnCl 550 mM
Ligand 100 uM
PEG 0.06 %
Glycerol 20 %
Salt MIX 264/11 mM Na/K
BSA 10 mg/mL
Reducing agent 5 mM


stock solutions we will need:


References

  1. http://www.piercenet.com/files/1453as4.pdf

    [Pierce]
  2. Vincentelli R, Canaan S, Campanacci V, Valencia C, Maurin D, Frassinetti F, Scappucini-Calvo L, Bourne Y, Cambillau C, and Bignon C. High-throughput automated refolding screening of inclusion bodies. Protein Sci. 2004 Oct;13(10):2782-92. DOI:10.1110/ps.04806004 | PubMed ID:15388864 | HubMed [Vincentelli]
  3. Willis MS, Hogan JK, Prabhakar P, Liu X, Tsai K, Wei Y, and Fox T. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions. Protein Sci. 2005 Jul;14(7):1818-26. DOI:10.1110/ps.051433205 | PubMed ID:15937284 | HubMed [Willis]

All Medline abstracts: PubMed | HubMed


|}


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