User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13: Difference between revisions
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==Designing Our Matrix== | ==Designing Our Matrix== | ||
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The problem with any | 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> | ||
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This excludes:<BR><BR> | This excludes:<BR><BR> | ||
<BR>all Non Detergent SulfoBetaines (NDSB's) that can or cannot be important | <BR>all Non Detergent SulfoBetaines (NDSB's) that can or cannot be important | ||
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7.5 HEPES<BR> | 7.5 HEPES<BR> | ||
8.2 TRIS<BR> | 8.2 TRIS<BR> | ||
9. | 9.2 CHES<BR> | ||
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I replaced BORATE pH 9.5 from <cite>Willis</cite> 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> | I replaced BORATE pH 9.5 from <cite>Willis</cite> 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> | ||
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<u>detergents | <u>detergents</u><BR> | ||
triton X 100<BR> | triton X 100<BR> | ||
tween 80<BR> | tween 80<BR> | ||
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<BR>''NB: NONE is a category for all subsequent categories, because it must be included for matrix generation'' | <BR>''NB: NONE is a category for all subsequent categories, because it must be included for matrix generation'' | ||
<BR><BR> | <BR><BR> | ||
<u>reducing agents | <u>reducing agents</u><BR> | ||
DTT<BR> | DTT<BR> | ||
TCEP<BR> | TCEP<BR> | ||
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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 | 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> | <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) | 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) | ||
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| Reducing agent||5||mM|| | | Reducing agent||5||mM|| | ||
|- | |- | ||
| | | Divalent cation||2||mM|| | ||
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|} | |} | ||
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|- | |- | ||
| Reducing agent||500||mM | | Reducing agent||500||mM | ||
|- | |||
| Divalent cation||200||mM | |||
|- | |- | ||
| | | | ||
|} | |} | ||
==Buffers== | ==Buffers== | ||
{| {{table}} | {| {{table}} |
Revision as of 21:00, 13 October 2009
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In the beginning there was Jackgetting 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 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.
Designing Our Matrix
additionally, 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. Stock Solutionsit 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.
NB: I used the totally wicked excel2wiki converter for the table code
Buffers
salt mix
divalent cation mix
detergents
ligand
amino acids
reducing agentsNB: stocks will be prepared without reducing agents! these should be added fresh every time
Matrixthe first matrix can be found here The above conditions were paired in "Custom Design" of JMP-IN as follows: pH: 5 level Categorical
References
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