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

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=Matrix=
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the first matrix  can be found [http://openwetware.org/wiki/User:Timothy_L._Foley/Notebook/refolding_matrix/2009/10/13/Refolding_Matrix-10-13-2009 here]
The above conditions were paired in "Custom Design" of JMP-IN as follows:
The above conditions were paired in "Custom Design" of JMP-IN as follows:
pH: 5 level Categorical
pH: 5 level Categorical

Revision as of 20:37, 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

divalent cation mix

divalent cations ' ' stock ' vol tot ' vol '
MgCl2200mM1M25mL5mL
CaCl210mM1M25mL0.25mL
total5.25mL
vol water19.75mL

detergents

detergents stock desired concentration stock (mL) water (mL)
TritonX25%6%25mL619
Tween 8020%6%25mL7.517.5
CHAPS6%25mL1.5 gto 25

ligand

ligand stock ' desired ' vol ' stock (uL) ' water '
Ligand A20mM1mM10mL500uL9.5mL

amino acids

amino acid mw ' stock ' vol ' mass
L-glutamate147g/mol5.5M50mL40.425
L-arginine211g/mol5.5M50mL58.025

reducing agents

NB: stocks will be prepared without reducing agents! these should be added fresh every time

reducing agents mw ' stock ' vol ' mass '
DTT155g/mol0.5M10mL0.775g
TCEP250g/mol0.5M10mL1.25g

Matrix

the first matrix can be found here The above conditions were paired in "Custom Design" of JMP-IN as follows: pH: 5 level Categorical

Buffer # pH detergent amino acids salt crowders divalent cation GnCl BSA Ligand reducing agent Y
15.5 MESnonenonelowPEG1010DTT
25.5 MESnonenonehighnone0000DTT
35.5 MESnonenonehighPEG0001none
45.5 MESnoneArgnonePEG1001TCEP
55.5 MESnoneGlutlowGlycerol1010TCEP
65.5 MESTritonXArgnoneGlycerol1011none
75.5 MESTritonXArghighnone0010TCEP
85.5 MESTritonXGlutnonePEG0010DTT
95.5 MESTritonXGlutlownone0101DTT
105.5 MESTritonXGlutlowGlycerol0101none
115.5 MESTween80nonelowGlycerol1110TCEP
125.5 MESTween80Arglownone0000DTT
135.5 MESTween80ArghighGlycerol0111TCEP
145.5 MESTween80GlutnoneGlycerol1101TCEP
155.5 MESTween80GluthighPEG0111none
165.5 MESCHAPSnonelownone0100none
175.5 MESCHAPSnonehighnone0111TCEP
185.5 MESCHAPSnonehighnone1101DTT
195.5 MESCHAPSArgnonePEG1000none
205.5 MESCHAPSGlutnoneGlycerol1110DTT
216.5 MESnonenonenonenone1001none
226.5 MESnonenonenoneGlycerol0110DTT
236.5 MESnoneArglownone1111none
246.5 MESnoneArghighPEG1110none
256.5 MESnoneGlutlowGlycerol1011DTT
266.5 MESTritonXnonenoneGlycerol1000TCEP
276.5 MESTritonXArglowPEG0111DTT
286.5 MESTritonXArghighGlycerol0000DTT
296.5 MESTritonXGlutlownone1100TCEP
306.5 MESTritonXGluthighGlycerol1101none
316.5 MESTween80nonenonenone0111DTT
326.5 MESTween80nonelowPEG1000TCEP
336.5 MESTween80nonelowPEG1001DTT
346.5 MESTween80ArgnonePEG0110TCEP
356.5 MESTween80Gluthighnone0010DTT
366.5 MESCHAPSnonehighGlycerol0110TCEP
376.5 MESCHAPSArghighPEG1110none
386.5 MESCHAPSArghighGlycerol0011none
396.5 MESCHAPSGlutnonenone1101none
406.5 MESCHAPSGlutlowPEG0001TCEP
417.5 HEPESnonenonelowPEG0101none
427.5 HEPESnoneArghighnone0011TCEP
437.5 HEPESnoneArghighGlycerol0100none
447.5 HEPESnoneGlutnonePEG0100TCEP
457.5 HEPESnoneGluthighGlycerol0000DTT
467.5 HEPESTritonXnonenonenone0011none
477.5 HEPESTritonXnonenonenone1010TCEP
487.5 HEPESTritonXArglowPEG0001TCEP
497.5 HEPESTritonXArglowGlycerol1110none
507.5 HEPESTritonXGluthighPEG1100TCEP
517.5 HEPESTween80nonenoneGlycerol1011none
527.5 HEPESTween80nonelowPEG0111DTT
537.5 HEPESTween80nonelowPEG1111TCEP
547.5 HEPESTween80Glutnonenone1111DTT
557.5 HEPESTween80Gluthighnone1001DTT
567.5 HEPESCHAPSnonelowGlycerol0100DTT
577.5 HEPESCHAPSArgnoneGlycerol1100TCEP
587.5 HEPESCHAPSArglownone0011none
597.5 HEPESCHAPSGluthighPEG1010DTT
608.2 TrisnonenonehighPEG0011TCEP
618.2 TrisnoneArgnonenone1110DTT
628.2 TrisnoneGlutnonenone0011none
638.2 TrisnoneGlutlownone0100TCEP
648.2 TrisnoneGlutlowGlycerol1110DTT
658.2 TrisTritonXnonelowPEG1111none
668.2 TrisTritonXnonehighGlycerol1010none
678.2 TrisTritonXArghighPEG1101DTT
688.2 TrisTritonXGluthighPEG0111TCEP
698.2 TrisTween80nonehighnone1100none
708.2 TrisTween80ArglowGlycerol0100DTT
718.2 TrisTween80ArghighGlycerol1001TCEP
728.2 TrisTween80GlutnoneGlycerol0001none
738.2 TrisCHAPSnonelownone1000none
748.2 TrisCHAPSnonelowGlycerol0011TCEP
758.2 TrisCHAPSArgnonenone0100DTT
768.2 TrisCHAPSArgnonePEG1001DTT
778.2 TrisCHAPSGlutnonePEG0010TCEP
789.2 CHESnonenonenoneGlycerol1101TCEP
799.2 CHESnoneArgnonePEG0110none
809.2 CHESnoneArglownone1101TCEP
819.2 CHESnoneGluthighGlycerol1001DTT
829.2 CHESTritonXnonenonenone0000TCEP
839.2 CHESTritonXnonenoneGlycerol0111DTT
849.2 CHESTritonXnonehighPEG1010DTT
859.2 CHESTritonXArglownone1111DTT
869.2 CHESTritonXGlutlownone0000none
879.2 CHESTween80nonehighPEG0100none
889.2 CHESTween80Arglownone1010TCEP
899.2 CHESTween80ArglowGlycerol0000none
909.2 CHESTween80GlutnonePEG0100none
919.2 CHESTween80Gluthighnone1010none
929.2 CHESCHAPSnonehighGlycerol1101DTT
939.2 CHESCHAPSArgnonePEG0001DTT
949.2 CHESCHAPSGlutlowPEG1010none
959.2 CHESCHAPSGlutlowGlycerol0011TCEP
969.2 CHESCHAPSGluthighnone1111TCEP


or the following matrix with the amino acids as a 4-factor categorical with none, glu, arg, glu+arg

buffer # pH detergent amino acids salt crowders divalent cation GnCl BSA Ligand reducing agent Amino acid
15.5 MESnonenonelownone1011noneArg
25.5 MESnonenonehighGlycerol0111TCEPGlu
35.5 MESnoneGlutnonenone0000noneArg+Glu
45.5 MESnoneGlutlowGlycerol0000TCEPnone
55.5 MESTritonXnonehighnone1001noneGlu
65.5 MESTritonXArglowPEG1111noneArg+Glu
75.5 MESTritonXArglowGlycerol0100TCEPArg
85.5 MESTritonXArghighGlycerol1110noneArg+Glu
95.5 MESTritonXGlutnonenone1010TCEPArg+Glu
105.5 MESTween80nonenonePEG0111DTTnone
115.5 MESTween80nonenoneGlycerol1100DTTArg
125.5 MESTween80ArgnonePEG0001TCEPArg
135.5 MESTween80ArglowGlycerol0000noneGlu
145.5 MESTween80Arghighnone1111DTTArg
155.5 MESCHAPSnonehighGlycerol0000DTTArg+Glu
165.5 MESCHAPSArgnonenone1001TCEPnone
175.5 MESCHAPSGlutnonePEG0101noneGlu
185.5 MESCHAPSGlutlowPEG1110DTTGlu
195.5 MESCHAPSGluthighPEG1110DTTnone
206.5 MESnonenonenonenone1100noneArg
216.5 MESnonenonelowGlycerol1001DTTnone
226.5 MESnoneArgnonePEG1001DTTnone
236.5 MESnoneGluthighnone0110noneArg+Glu
246.5 MESnoneGluthighPEG0100TCEPArg+Glu
256.5 MESTritonXnonelownone0100TCEPnone
266.5 MESTritonXnonehighGlycerol1111DTTArg
276.5 MESTritonXArgnonenone0100noneGlu
286.5 MESTritonXArghighPEG1011DTTArg+Glu
296.5 MESTritonXGlutlowGlycerol1011noneArg
306.5 MESTween80Argnonenone0111DTTArg
316.5 MESTween80ArgnoneGlycerol0011noneArg+Glu
326.5 MESTween80GlutlowPEG1100TCEPGlu
336.5 MESTween80GlutlowGlycerol1111TCEPnone
346.5 MESTween80GluthighGlycerol0111noneGlu
356.5 MESCHAPSnonenonenone0010TCEPArg
366.5 MESCHAPSnonelowPEG0010noneGlu
376.5 MESCHAPSnonelowGlycerol1000TCEPArg+Glu
386.5 MESCHAPSArghighPEG0001TCEPnone
396.5 MESCHAPSArghighPEG1000DTTGlu
407.5 HEPESnonenonehighnone1001TCEPGlu
417.5 HEPESnoneArgnoneGlycerol0010DTTGlu
427.5 HEPESnoneGlutnoneGlycerol1111TCEPArg+Glu
437.5 HEPESnoneGlutlownone0011DTTnone
447.5 HEPESnoneGlutlowPEG0001DTTArg
457.5 HEPESTritonXnonenonePEG1110DTTGlu
467.5 HEPESTritonXnonelownone0101DTTGlu
477.5 HEPESTritonXnonehighPEG0011noneArg
487.5 HEPESTritonXArgnonePEG0010nonenone
497.5 HEPESTritonXGluthighGlycerol0000TCEPGlu
507.5 HEPESTween80nonelowPEG1011TCEPArg+Glu
517.5 HEPESTween80nonehighGlycerol1010TCEPArg+Glu
527.5 HEPESTween80Arghighnone1000DTTnone
537.5 HEPESTween80GlutnonePEG1100TCEPArg+Glu
547.5 HEPESTween80Gluthighnone1100noneArg
557.5 HEPESCHAPSnonenonenone0111nonenone
567.5 HEPESCHAPSArgnoneGlycerol1100nonenone
577.5 HEPESCHAPSArglownone0110DTTArg+Glu
587.5 HEPESCHAPSArglowGlycerol1101TCEPArg
597.5 HEPESCHAPSGluthighGlycerol0101noneArg
608.2 TrisnonenonenonePEG0100TCEPArg
618.2 TrisnonenonenoneGlycerol0110nonenone
628.2 TrisnoneArghighnone1111TCEPGlu
638.2 TrisnoneArghighPEG0101noneArg+Glu
648.2 TrisnoneArghighPEG1000DTTArg
658.2 TrisTritonXnonelowPEG0001noneArg+Glu
668.2 TrisTritonXGlutnonePEG1110TCEPArg
678.2 TrisTritonXGlutnoneGlycerol1011TCEPnone
688.2 TrisTritonXGlutlownone1100DTTnone
698.2 TrisTritonXGluthighGlycerol0001DTTnone
708.2 TrisTween80nonelownone0010DTTArg
718.2 TrisTween80nonehighnone0000nonenone
728.2 TrisTween80ArglowPEG0111TCEPGlu
738.2 TrisTween80Arghighnone1010TCEPArg+Glu
748.2 TrisCHAPSnonenoneGlycerol1101DTTArg+Glu
758.2 TrisCHAPSArglowGlycerol0110DTTGlu
768.2 TrisCHAPSGlutnonenone1011noneGlu
778.2 TrisCHAPSGlutlowGlycerol1001noneArg
789.2 CHESnonenonenoneGlycerol1010DTTGlu
799.2 CHESnoneArglownone0101TCEPArg+Glu
809.2 CHESnoneArglowPEG1010noneArg
819.2 CHESnoneArglowGlycerol1110nonenone
829.2 CHESnoneGluthighnone1111DTTGlu
839.2 CHESTritonXnonehighGlycerol0110TCEPnone
849.2 CHESTritonXArgnonenone1001TCEPGlu
859.2 CHESTritonXArghighGlycerol0100DTTArg
869.2 CHESTritonXGlutnonePEG0101DTTArg+Glu
879.2 CHESTween80nonelowPEG1101nonenone
889.2 CHESTween80nonehighPEG1100noneGlu
899.2 CHESTween80ArgnoneGlycerol1001noneGlu
909.2 CHESTween80GlutnoneGlycerol0001DTTArg+Glu
919.2 CHESTween80Glutlownone0010DTTArg+Glu
929.2 CHESCHAPSnonelownone1101DTTArg+Glu
939.2 CHESCHAPSnonehighPEG0011TCEPArg
949.2 CHESCHAPSArghighnone0111TCEPnone
959.2 CHESCHAPSGlutnonenone0010TCEPArg
969.2 CHESCHAPSGluthighPEG1010nonenone

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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