6. Assays for membrane proteins
•Overexpressed recombinant catalytic fragments
•Assay in solution; Highly disordered
7. Assays for membrane proteins
• All cellular proteins present (complex)
• Biologically relevant context
• Laborious, slow, costly
8. Assays for membrane proteins
Is there an assay system that:
•Is simple to use?
•Compatible with HTS?
•Imparts the organization and
context of a biological
membrane?
YES!
11. Template Directed Assembly
•HIS-tagged proteins adhere to fully fluid surface
•Surface dictates biological organization:
•topology and conformation
•relationship to other subunits
12. Template Directed Assembly
•HIS-tagged proteins adhere to fully fluid surface
•Surface dictates biological organization:
•topology and conformation
•relationship to other subunits
•Promotes self-assembly:
•homo- and hetero- multimers
Fluid surface
Functional
Assay
13. Template Directed Assembly
•HIS-tagged proteins adhere to fully fluid surface
•Surface dictates biological organization:
•topology and conformation
•relationship to other subunits
•Promotes self-assembly:
•homo- and hetero- multimers
•higher-order complexes
Fluid surface
Functional
Assay
14. Functional dimerization of Insulin RTK
•Insulin receptor is normally activated through
addition of high levels of manganese
•HIS-tagged InR intracellular domain presented in
context of TDA 2.0™ dimerizes and trans-
phosphorylates in normal salt and Mn++
P- -P P- -P
Template
“First-Step”
15. Functional dimerization of Insulin RTK
•Insulin receptor is normally activated through
addition of high levels of manganese
•HIS-tagged InR intracellular domain presented in
context of TDA 2.0™ dimerizes and trans-
phosphorylates in normal salt and Mn++
P- -P
7-fold increase in
activity towards
IRS1-derived peptide
substrate!
Template Template
16. TDA 2.0™ affect on other membrane kinases
200 100 50 25 10 5 1
[Enzyme] (nM)
17. Membrane context confers altered,
relevant, substrate selectivity
•TrkB was assayed against a random peptide
library in the absence and presence of TDA 2.0™
• Substrate selection was remarkably different
• Known substrates were only found in the
presence of TDA 2.0™
Y1173 EGFR
Y939 IRS1
Y987 IRS1 Y612 IRS1
18. Assembly of multi-component complexes
•Significant literature suggesting that “accessory”
proteins greatly affect enzyme pharmacology
• TDA 2.0™ offers a simple way to assess enzyme
activity in the proper context
Insulin/IGF-1 Receptor EGF Receptor EGF/Her2
19. Assembly of multi-component complexes
•Significant literature suggesting that “accessory”
proteins greatly affect enzyme pharmacology
• TDA 2.0™ offers a simple way to assess enzyme
activity in the proper context
Insulin/IGF-1 Receptor EGF Receptor EGF/Her2
20. Assembly of multi-component complexes
•Significant literature suggesting that “accessory”
proteins greatly affect enzyme pharmacology
• TDA 2.0™ offers a simple way to assess enzyme
activity in the proper context
Insulin/IGF-1 Receptor EGF Receptor EGF/Her2
22. Want TDA 2.0™ in your lab?
TDA my way!
Our SmartScreen™ kits contain pre- Our field applications scientific team (FAST)
validated enzymes optimized with TDA 2.0™ can help implement TDA 2.0™ technology in
your lab, or develop a customized assay to
suit your application!
www.blueskybiotech.com
products@blueskybiotech.com
