User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13: Difference between revisions
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= | =Refolding Matrix design and use= | ||
getting started here... The background reading I have done that will weigh heavy today: | getting started here... The background reading I have done that will weigh heavy today: | ||
The Pierce Refolding Kit Instructions<cite>Pierce</cite> that detail the 9 buffer components in their kit, which are: | The Pierce Refolding Kit Instructions<cite>Pierce</cite> that detail the 9 buffer components in their kit, which are: | ||
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=Matrix= | =Matrix= | ||
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 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:<br> | ||
pH: 5 level Categorical | pH: 5 level Categorical<br> | ||
Detergent: 4 categories (none, triton, tween, chaps) | Detergent: 4 categories (none, triton, tween, chaps)<br> | ||
amino acids: 3 categories (none, arg, | amino acids: 3 categories (none, arg, glu)<br> | ||
salts: none low high | salts: none low high<br> | ||
crowders: none PEG Glycerol | crowders: none PEG Glycerol <br> | ||
divalent cation: 0, 1 <br> | divalent cation: 0, 1 <br> | ||
GnCl: : 0, 1 <br> | GnCl: : 0, 1 <br> | ||
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the second matrix matrix can be found [http://openwetware.org/wiki/User:Timothy_L._Foley/Notebook/refolding_matrix/2009/10/13/Refolding_Matrix-10-13-2009_amino_acids_4-factor here] | the second matrix matrix can be found [http://openwetware.org/wiki/User:Timothy_L._Foley/Notebook/refolding_matrix/2009/10/13/Refolding_Matrix-10-13-2009_amino_acids_4-factor here] | ||
or the following matrix with the amino acids as a 4-factor categorical with none, glu, arg, glu+arg | or the following matrix with the amino acids as a 4-factor categorical with (none, glu, arg, glu+arg) | ||
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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.. | |||
----<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.''' | |||
==References== | ==References== | ||
<biblio> | <biblio> |
Revision as of 10:30, 25 January 2011
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Refolding Matrix design and usegetting 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:
it took all afternoon to prep the stock solutions above. Neither pH meter probe works, and we do not have any solid TCEP. References
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