Optimum Solubility Screen
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The work described here was supported by the National Institutes of Health GM 62412 PowerPoint PPT Presentation


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Optimum Solubility Screen To optimize buffer conditions for homogeneity and crystallization of proteins Jarmila Jancarik, Ramona Pufan, Connie Hong and Rosalind Kim Lawrence Berkeley National Laboratory PPCW, March 29-31, 2004.

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The work described here was supported by the National Institutes of Health GM 62412

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The work described here was supported by the national institutes of health gm 62412

Optimum Solubility Screen

To optimize buffer conditions for homogeneity and crystallization of proteins

Jarmila Jancarik, Ramona Pufan, Connie Hong and Rosalind Kim

Lawrence Berkeley National Laboratory

PPCW, March 29-31, 2004

The work described here was supported by the National Institutes of Health GM 62412


Step 1 optimium solubility screen

Step 1Optimium Solubility Screen

Aggregated protein in purification buffer

1 µl

Buffer

1 µl

Protein

Set up protein using 24-well Linbro plates,

use 0.5 ml buffer (24) in reservoir; 1 µl buffer + 1 µl protein

on the cover slip (vapor diffusion method)

Incubate overnight over the same buffer

buffer

Perform Dynamic Light Scattering (DLS) on clear drops, pick best condition.


The work described here was supported by the national institutes of health gm 62412

Linbro Plate

List of optimization buffers

Version 1


The work described here was supported by the national institutes of health gm 62412

Step 2

Additive Screen

Test additives using best buffer:

25, 50, 100 mM NaCl

5 and 10% glycerol

2 mM CHAPS (CMC=6 to 10 mM)

0.1%, 1% Octylglucoside (CMC=0.53%)

0.1%, 1% Dodecyl Maltoside (CMC=0.0087%)

30mM TCEP, 5mM DTT,10 mM β-mercaptoethanol

Two hour incubation

Perform DLS

Pick best additive, exchange protein into optimal buffer/additive

Set up Crystal Screens


Dls in different buffers

DLS in Different Buffers

1371B in Buffer #16

100 mM Na K Phosphate, pH 7.0

1371B in Buffer #21

100 mM Tris, pH 8.5


1371b

1371B

1371B in 100 mM Tris, pH 8.5

1% Octylglucoside

Crystals of 1371B

Structure solved


The work described here was supported by the national institutes of health gm 62412

Results

* Radius in nanometers, % polydispersity

**Crystals appeared in Optimium Screen


Summary

Summary

  • This assay provides an opportunity to find an optimum buffer/additive for crystallization of a given protein.

  • Screen is fast and requires a small amount of protein.

  • Out of 14 targets, protein monodispersity improved greatly for 11 targets and 9 of these proteins crystallized.

  • This screen is being implemented into a high throughput protocol.


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