Families of membrane proteins
•Single- and multi-pass transmembrane proteins
Families of membrane proteins
       •Single- and multi-pass transmembrane proteins
       •Peripheral membrane proteins

...
Families of membrane proteins
       •Single- and multi-pass transmembrane proteins
       •Peripheral membrane proteins
 ...
Families of membrane proteins
                     •Single- and multi-pass transmembrane proteins
                     •Pe...
Assays for membrane proteins
•Overexpressed recombinant catalytic fragments
•Assay in solution; Highly disordered
Assays for membrane proteins
• All cellular proteins present (complex)
       • Biologically relevant context
• Laborious,...
Assays for membrane proteins




Is there an assay system that:
       •Is simple to use?
    •Compatible with HTS?
•Impar...
Assays for membrane proteins
Template Directed Assembly




                      +




His-tagged Proteins
Template Directed Assembly
•HIS-tagged proteins adhere to fully fluid surface
•Surface dictates biological organization:
 ...
Template Directed Assembly
•HIS-tagged proteins adhere to fully fluid surface
•Surface dictates biological organization:
 ...
Template Directed Assembly
•HIS-tagged proteins adhere to fully fluid surface
•Surface dictates biological organization:
 ...
Functional dimerization of Insulin RTK
           •Insulin receptor is normally activated through
           addition of h...
Functional dimerization of Insulin RTK
           •Insulin receptor is normally activated through
           addition of h...
TDA 2.0™ affect on other membrane kinases




200   100   50    25     10   5   1
             [Enzyme] (nM)
Membrane context confers altered,
                             relevant, substrate selectivity
                           ...
Assembly of multi-component complexes
                                •Significant literature suggesting that “accessory”
...
Assembly of multi-component complexes
                                •Significant literature suggesting that “accessory”
...
Assembly of multi-component complexes
                                •Significant literature suggesting that “accessory”
...
Assembly of multi-component complexes
                                             •Activation of Akt by mTOR and PDK1 on ...
Want TDA 2.0™ in your lab?



                                                           TDA my way!
    Our SmartScreen™ ...
Smart screen animations_Blue Sky Biotech
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Smart screen animations_Blue Sky Biotech

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Blue Sky's field applications scientific team (FAST) can help implement TDA 2.0™ technology in your lab, or develop a customized assay to suit your application!

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Smart screen animations_Blue Sky Biotech

  1. 1. Families of membrane proteins •Single- and multi-pass transmembrane proteins
  2. 2. Families of membrane proteins •Single- and multi-pass transmembrane proteins •Peripheral membrane proteins Membrane Associated
  3. 3. Families of membrane proteins •Single- and multi-pass transmembrane proteins •Peripheral membrane proteins •Lipid-anchored (Farnesylated, prenylated, etc.) Membrane Associated Membrane Targeted
  4. 4. Families of membrane proteins •Single- and multi-pass transmembrane proteins •Peripheral membrane proteins •Lipid-anchored (Farnesylated, prenylated, etc.) Membrane Associated Membrane Targeted ?? Cytoplasmic
  5. 5. Assays for membrane proteins •Overexpressed recombinant catalytic fragments •Assay in solution; Highly disordered
  6. 6. Assays for membrane proteins • All cellular proteins present (complex) • Biologically relevant context • Laborious, slow, costly
  7. 7. 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!
  8. 8. Assays for membrane proteins
  9. 9. Template Directed Assembly + His-tagged Proteins
  10. 10. Template Directed Assembly •HIS-tagged proteins adhere to fully fluid surface •Surface dictates biological organization: •topology and conformation •relationship to other subunits
  11. 11. 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
  12. 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 •higher-order complexes Fluid surface Functional Assay
  13. 13. 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”
  14. 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 7-fold increase in activity towards IRS1-derived peptide substrate! Template Template
  15. 15. TDA 2.0™ affect on other membrane kinases 200 100 50 25 10 5 1 [Enzyme] (nM)
  16. 16. 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
  17. 17. 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
  18. 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. 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. 20. Assembly of multi-component complexes •Activation of Akt by mTOR and PDK1 on TDA 2.0™ pmol Phosphate Added 1 2 3 4 5 6 7 8 9 10 mTOR Anti-GST PDK1 Akt1 Anti-HIS Phospho-Akt1 Anti-Akt p-S473 TDA 2.0™ - + - - - + - + - + Akt1 + + - - + + + + + + Phospho-Akt1 Anti-Akt p-T308 PDK1 - - + - + + - - + + mTOR - - - + - - + + + +
  21. 21. 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
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