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

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Revision as of 15:12, 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)

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
none

additives:
arginine/asparate/none
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
Buffer10X10
detergent100X1
amino acids5X20
GnCl10X10
Ligand10X10
PEG100X1
Glycerol5X20
Salt MIX10X10
BSA10X10
Reducing agent100X1
total93

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

so we are set.

component concentrations from [3] units interconversion
Buffer50mM
detergent0.5mM~0.06%
amino acids550mM
GnCl550mM
Ligand100uM
PEG0.06%
Glycerol20%
Salt MIX264/11mM Na/K
BSA10mg/mL
Reducing agent5mM


stock solutions we will need:


stock solution concentration units
Buffer500mM
detergent6%
amino acids5500mM
GnCl5500mM
Ligand1000uM
PEG6%
Glycerol100%
Salt MIX2640/110mM Na/K
BSA100mg/mL
Reducing agent500mM

Buffers

Buffer molecular weight stock solution volume mass '
MES195500504.875g
HEPES238.3500505.9575g
TRIS121.1500503.0275g
CHES207.3500505.1825g

salt mix

high salt component stock conc units desired conc vol units volumes '
NaCl5M2.6450mL26.4mL
KCl4M0.1150mL1.375mL
water22.225mL
low salt
NaCl5M0.26450mL2.64mL
KCl4M0.01150mL0.1375mL
water47.2225mL

detergents

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. . pmid:15388864. PubMed HubMed [Vincentelli]
  3. Willis MS, Hogan JK, Prabhakar P, Liu X, Tsai K, Wei Y, and Fox T. . pmid:15937284. PubMed HubMed [Willis]
All Medline abstracts: PubMed HubMed



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