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

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I am partial to exploring the largest number of possible buffer configurations, but I want it to still be worth while, i.e. if one of these randomized ingredients  has negative effects on folding, a large portion of the wells is worthless.
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I am partial to exploring the largest number of possible buffer configurations, but I want it to still be worth while, i.e. if one of these randomized ingredients  has negative effects on folding, a large portion of the wells is worthless. The software wants to prepare 480 wells as a default for a single iteration of the experiment... this is too many... I set the value at 96..
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Revision as of 00:33, 14 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 report on buffer matricies is that they are packed full of miscellaneous chemicals that are either not in house, or prohibitively expensive... it is my experience (professor management theory) that the best way to get expensive materials/equpment to support a new method/tehcnique is to furnish results that are desired but were only accessible from that new method/technique (grunt work), and then ask for $$$ chemicals to improve future experiments...
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
9.2 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
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
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)


Every additive looks reasonable except for 1... BSA.
And, 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 the total volume for the experiment...and that all components are soluble at the concentration determined here for the 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)

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
Divalent cation2mM


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
Divalent cation200mM

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
Detergent: 4 categories (none, triton, tween, chaps)
amino acids: 3 categories (none, arg, glu)
salts: none low high
crowders: none PEG Glycerol
divalent cation: 0, 1
GnCl: : 0, 1
BSA: 0, 1
Ligand: 0, 1
Reducing agent: 0, 1


the second matrix matrix can be found here or the following matrix with the amino acids as a 4-factor categorical with (none, glu, arg, glu+arg)

I am partial to exploring the largest number of possible buffer configurations, but I want it to still be worth while, i.e. if one of these randomized ingredients has negative effects on folding, a large portion of the wells is worthless. The software wants to prepare 480 wells as a default for a single iteration of the experiment... this is too many... I set the value at 96..




it took all afternoon to prep the stock solutions above. Neither pH meter probe works, and we do not have any solid TCEP.
I will go to the Core tomorrow and acquire some fresh DTT and TCEP.
NOTE: both amino acid solutions were adjusted to be 1/2 the concentration here. 58g Arg in 50 mL water (116% w/v solution) seemed a bit over the top.

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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