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96 wells stability optimization screens

 

 

This is based on two ready-made 96-condition screens developed by our colleagues at EMBL Hamburg (See Boivin et al. Protein Expr Purif (2013). 91, 192–206 and below). 

 

 If you want to perform TSA optimization please contact us at This email address is being protected from spambots. You need JavaScript enabled to view it. 

 

Practical information

 

  • 210ul of pure protein at 20-100uM is required to performed one 96-condition screen
  • Protein-Protein complexes may lead to curves that are difficult to interpret. We recommend running our standard TSA protocol first.
  • Often lipids interfere with TSA experiment. We recommend running our standard TSA protocol first.
  • For the SPC 2 screen, 10mL of the buffer condition you want to use as a base and 5ul for each of your own additives at 5X concentration are required.

 

Description of the ready-made screens.

 

RUBIC “Buffer screen”

 

This screen focuses on identification of buffer conditions improving your sample stability through a matrix exploring 4 different parameters: buffer type, buffer concentration, pH and salt concentration:

  • 14 different buffers
  • Buffer concentration: 0.02M to 0.25M
  • pH: 4 to 10
  • NaCl concentration: 0 - 1M

The formulation of the screen is available here: RUBIC buffer screen

 

tsa_1Figure 1: Stabilization effect of 14 different buffers (normalized data). The red curve indicates a stabilization effect, the green curve indicates decreased stability

 

SPC 2 “Additives screen”

 

This screen is designed to test the effect of 76 additives under one constant buffer condition. We recommend to perform this screen after the SPC1 screen. The additives include the following categories:

  • Monovalent ions
  • Co-factors
  • Polyols
  • Multivalent ions
  • Carbohydrates
  • Salts
  • Amino Acids
  • Reducing reagents
  • Linker
  • Polyamines
  • Chaotropic reagents
  • Detergents
  • Chelating reagent
  • Dissociation reagents


The screen contains 6 free wells that can be use to test your specific additives.

The formulation of the screen is available here: SPC 2 description

 

tsa_2
Fig 2: Stabilization effect of a co-factor on a protein kinase (Green curve, normalized data)

 

Data analysis

Data analysis can be performed directly through the CRIMS thermofluor interface.

tsa_menu
Figure 3: The thermofluor experiments interface in CRIMS

 

The thermofluor CRIMS interface allows the user:

  • To calculate the melting temperature (Tm) for each condition
  • To display raw data and normalized data for analysis
  • To directly focus on conditions that belong to the same analyzed parameter (i.e.: buffer) through tabs
  • To store interesting conditions into a summary for further analysis

 

 

 

References

Boivin et al. Protein Expr Purif (2013). 91, 192–206