# User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13

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 Revision as of 15:42, 13 October 2009 (view source)← Previous diff Revision as of 15:46, 13 October 2009 (view source)Next diff → Line 91: Line 91:

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}} +

+ + {| {{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 128: Line 130: {| {{table}} {| {{table}} | align="center" style="background:#f0f0f0;"|'''component''' | align="center" style="background:#f0f0f0;"|'''component''' - | align="center" style="background:#f0f0f0;"|'''concentrations from Willis''' + | align="center" style="background:#f0f0f0;"|'''concentrations from Willis''' - | align="center" style="background:#f0f0f0;"|'''''' + | align="center" style="background:#f0f0f0;"|'''units''' - | align="center" style="background:#f0f0f0;"|'''''' + | align="center" style="background:#f0f0f0;"|'''interconversion''' |- |- | Buffer||50||mM|| | Buffer||50||mM|| Line 159: Line 161: + {| {{table}} + | align="center" style="background:#f0f0f0;"|'''stock solution''' + | align="center" style="background:#f0f0f0;"|'''concentration''' + | align="center" style="background:#f0f0f0;"|'''units''' + |- + | Buffer||500||mM + |- + | detergent||6||% + |- + | amino acids||5500||mM + |- + | GnCl||5500||mM + |- + | Ligand||1000||uM + |- + | PEG||6||% + |- + | Glycerol||100||% + |- + | Salt MIX||2640/110||mM Na/K + |- + | BSA||100||mg/mL + |- + | Reducing agent||500||mM + |- + | + |} ==References== ==References==

## Revision as of 15:46, 13 October 2009

Refolding Matrix Main project page
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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)

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

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)

 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 [3] units interconversion 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:

 stock solution concentration units Buffer 500 mM detergent 6 % amino acids 5500 mM GnCl 5500 mM Ligand 1000 uM PEG 6 % Glycerol 100 % Salt MIX 2640/110 mM Na/K BSA 100 mg/mL Reducing agent 500 mM

## References

Error fetching PMID 15388864:
Error fetching PMID 15937284:
1. http://www.piercenet.com/files/1453as4.pdf [Pierce]
2. Error fetching PMID 15388864: [Vincentelli]
3. Error fetching PMID 15937284: [Willis]
All Medline abstracts: PubMed HubMed