User:Timothy L. Foley/Notebook/refolding matrix/2009/10/13: Difference between revisions
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guanidinium chloride (GnCl)<BR> | guanidinium chloride (GnCl)<BR> | ||
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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! | |||
<BR><BR> | <BR><BR> | ||
----<BR> | ----<BR> | ||
A paper on High throughput automated refolding <cite>Vincentelli</cite> that describes a 96 well matrix of buffer conditions they developed for structural genomics projects for Mtb and Viruses. <BR> | A paper on High throughput automated refolding <cite>Vincentelli</cite> that describes a 96 well matrix of buffer conditions they developed for structural genomics projects for Mtb and Viruses. <BR> | ||
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<br> | |||
Finally, Vertex Pharmaceuticals <cite>Willis</cite> 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> | Finally, Vertex Pharmaceuticals <cite>Willis</cite> 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> | ||
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Why BSA? | Why BSA? this is my screen, and a I want to try something wacky.<BR> | ||
===Stock Solutions=== | ===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 | 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 | ||
<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) | ||
{| {{table}} | <BR><BR>{| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''component''' | | align="center" style="background:#f0f0f0;"|'''component''' | ||
| align="center" style="background:#f0f0f0;"|'''stock solution''' | | align="center" style="background:#f0f0f0;"|'''stock solution''' | ||
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|} | |} | ||
''NB: I used the totally wicked [http://excel2wiki.net/index.php excel2wiki] converter for the table code'' | ''NB: I used the totally wicked [http://excel2wiki.net/index.php excel2wiki] converter for the table code'' | ||
<BR><BR> | |||
so we are set. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''component''' | |||
| align="center" style="background:#f0f0f0;"|'''concentrations from Willis''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| Buffer||50||mM|| | |||
|- | |||
| detergent||0.5||mM||~0.06% | |||
|- | |||
| amino acids||550||mM|| | |||
|- | |||
| GnCl||550||mM|| | |||
|- | |||
| Ligand||100||uM|| | |||
|- | |||
| PEG||0.06||%|| | |||
|- | |||
| Glycerol||20||%|| | |||
|- | |||
| Salt MIX||264/11||mM Na/K|| | |||
|- | |||
| BSA||10||mg/mL|| | |||
|- | |||
| Reducing agent||5||mM|| | |||
|- | |||
| | |||
|} | |||
stock solutions we will need: | |||
Revision as of 12:42, 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
|
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 | |||
NB: I used the totally wicked excel2wiki converter for the table code
so we are set.
component | concentrations from Willis | ' | ' |
Buffer | 50 | mM | |
detergent | 0.5 | mM | ~0.06% |
amino acids | 550 | mM | |
GnCl | 550 | mM | |
Ligand | 100 | uM | |
PEG | 0.06 | % | |
Glycerol | 20 | % | |
Salt MIX | 264/11 | mM Na/K | |
BSA | 10 | mg/mL | |
Reducing agent | 5 | mM | |
stock solutions we will need:
References
- Vincentelli R, Canaan S, Campanacci V, Valencia C, Maurin D, Frassinetti F, Scappucini-Calvo L, Bourne Y, Cambillau C, and Bignon C. High-throughput automated refolding screening of inclusion bodies. Protein Sci. 2004 Oct;13(10):2782-92. DOI:10.1110/ps.04806004 |
- Willis MS, Hogan JK, Prabhakar P, Liu X, Tsai K, Wei Y, and Fox T. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions. Protein Sci. 2005 Jul;14(7):1818-26. DOI:10.1110/ps.051433205 |
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