User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13: Difference between revisions
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Why BSA? it just works for me.<BR> | Why BSA? it just works for me.<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 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 | |||
Buffer 10X 10 | |||
detergent 100X 1 | |||
amino acids 5X 20 | |||
GnCl 10X 10 | |||
Ligand 10X 10 | |||
PEG 100X 1 | |||
Glycerol 5X 20 | |||
Salt MIX 10X 10 | |||
BSA 10X 10 | |||
Reducing agent 100X 1 | |||
total 93 | |||
Revision as of 12:14, 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 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 Buffer 10X 10 detergent 100X 1 amino acids 5X 20 GnCl 10X 10 Ligand 10X 10 PEG 100X 1 Glycerol 5X 20 Salt MIX 10X 10 BSA 10X 10 Reducing agent 100X 1 total 93
References
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