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

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=Refolding Matrix design and use= getting started here... The background reading I have done that will weigh heavy today: The Pierce Refolding Kit Instructions Pierce 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 Vincentelli that describes a 96 well matrix of buffer conditions they developed for structural genomics projects for Mtb and Viruses. 

Finally, Vertex Pharmaceuticals Willis 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>

Designing Our Matrix
<BR> 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...<BR> This excludes:<BR><BR> <BR>all Non Detergent SulfoBetaines (NDSB's) that can or cannot be important <BR>a-cyclodextrin and methyl-B-cyclodextrin (NB: B-cyclodextrin that we have in house is not soluble in H2O.) <BR>fancy detergents (lauryl maltoside) <BR>N-lauryl sarcosine (iFOLD kits) <BR>FoldACEs (iFOLD series 3 kit) <BR>disco reducing agents (BMC: bis-mercaptoacetamide cyclohexane) <BR><BR>

additionally, <BR> <BR> From Willis' Willis report, buffer pH, detergent, and NDSB 201 were important for some proteins,<BR><BR>

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.<BR> <BR> The conditions we will screen follow:<BR> <BR> pH/ Buffer <BR> 5.5 MES<BR> 6.5 MES<BR> 7.5 HEPES<BR> 8.2 TRIS<BR> 9.2 CHES<BR> <BR> I replaced BORATE pH 9.5 from Willis 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).<BR><BR> <BR> detergents <BR> triton X 100<BR> tween 80<BR> Chaps<BR> none<BR> <BR>NB: NONE is a category for all subsequent categories, because it must be included for matrix generation <BR><BR> reducing agents <BR> DTT<BR> TCEP<BR> none<BR> <BR> additives :<BR> arginine/asparate/none<BR> divalent cations (2 Mg++/ 0.1 Ca++)<BR> GnCl<BR> Ligand<BR> PEG 3350/Glycerol/Sucrose<BR> BSA<BR> NaCl/KCl (high ionic strength)<BR> <BR> <BR> Every additive looks reasonable except for 1... BSA. <BR>And, why BSA? this is my screen, and a I want to try something wacky.<BR><BR>

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. <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) <BR><BR>

NB: I used the totally wicked excel2wiki converter for the table code <BR><BR> so we are set.

stock solutions we will need:

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

=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..

<BR><BR> it took all afternoon to prep the stock solutions above. Neither pH meter probe works, and we do not have any solid TCEP.<BR> I will go to the Core tomorrow and acquire some fresh DTT and TCEP.<BR> '''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.'''