Meso Scale Discovery Applications

Transcription

1 A Suite of Assays to Detect hosphorylated Receptor Tyrosine Kinases Associated with Neoplasia Timothy S. Schaefer, Jane Zhang, Sean Higgins, Laura K. Schaefer, Robert M. Umek and Jacob N. Wohlstadter Reversible tyrosine phosphorylation is a critical process in the transduction of signals from the cell surface to the nucleus resulting in global changes in gene expression. A common hallmark of most neoplasia is the inappropriate expression or activation of cell surface proteins possessing inherent tyrosine kinase activity. These membrane spanning receptor tyrosine kinases (RTKs) phosphorylate themselves on tyrosine residues located on the cytoplasmic portion of the protein that can serve as docking sites for adapter proteins. The resulting multiprotein complexes form the framework for numerous signaling pathways involved in proliferation, differentiation, angiogenesis and cell survival. Here we describe a suite of individual and multiplex assays designed to assess the level of phosphorylation of EGFR, ErbB, VEGFR-, DGFR- and c-kit in cellular lysates which can serve as surrogates to assess disease states. The assays are facile, sensitive and can be performed more rapidly than immunoblots and ELISAs and require considerably less material. Meso Scale Discovery Applications

2 Five RTKs and Signaling athways Associated with Neoplasia EGFR / ErbB EGFR Grb LC-G VEGFR- c-kit DGFR-B AK Vav ERK/ JAK Breast Head & Neck Lung Glioma Colon Shc p/p Activation of Kinase Activity Akt MAK/ERK Breast Gastric Ovarian Glioma Akt Solid Tumors (roangiogenesis) Myeloproliferation disorders Medulloblastoma Gastrointestinal Stromal Tumors

3 MSD MULTI-ARRAY TM Technology and MULTI-SOT lates Assay Format Electrochemiluminescent Reporter Antibody hosphoprotein in Cell Lysate Capture Antibody Electrode to Initiate Electrochemiluminescence General rotocol. MULTI-SOT Spot -Well lates precoated with capture antibodies are blocked with L of MSD Blocker A for hr with shaking, followed by washing.. L of cell lysates are added to the wells and incubated for hr with shaking, followed by washing.. L MSD SULFO-TAG TM -labeled antibodies (in antibody dilution buffer) are added to the wells and incubated for hr with shaking, followed by washing.. L MSD Read Buffer T (with surfactant) is added to the wells and analyzed on the SECTOR instrument.

4 Detection of hosphorylated and Total EGFR (ptyr) in the Same Well (A Cells),,,,,,,, ositive ative pegfr tegfr,,,,,,,,, EGFR ptyr MSD MULTI-SOT -Well -Spot late EGFR anti-egfr SULFO-TAG antibody ositive/ative cell lysate Capture antibody pegfr tegfr ,,,,,,,,,,,,,,,,, pegfr ositive,,,,,,,,,,,,,,,, pegfr ative,,,,,,, /N pegfr EGFR os g lysate per lane. g lysate per well Serum-deprived A cells were treated with Compound ( M;. hr)(negative) or EGF ( ng/ml; min)(positive). Whole cell lysates were added to MSD MULTI-SOT -Spot plates coated with capture antibody on two of the four spatially distinct electrodes per well. hosphorylated proteins were detected with MSD SULFO-TAG-labeled detection antibodies. os

5 Detection of hosphorylated EGFR (ptyr),,,,,,, ositive ative MSD MULTI-SOT -Well -Spot late A B anti-phospho-egfr SULFO-TAG antibody, ptyr ositive/ative cell lysate anti-egfr antibody, pegfr os os EGFR ,,,,,, pegfr ositive pegfr ative /N g lysate per lane. g lysate per well Serum-deprived A cells were treated with Compound ( M;. hr) (negative) or EGF ( ng/ml; min)(positive). Whole cell lysates were added to MSD MULTI-SOT -Spot plates coated with anti-total-egfr antibody on one of the four spatially distinct electrodes per well. hosphorylated EGFR was detected with anti-phospho-egfr antibody labeled with MSD SULFO-TAG reagent.

6 Detection of hosphorylated VEGFR- (ptyr/), ositive, ative,,, MSD MULTI-SOT -Well -Spot late A B anti-vegfr- SULFO-TAG antibody ositive/ative cell lysate anti-phospho-vegfr- antibody, ptyr/, os pvegfr- os pvegfr- ositive,,,,,,,,, pvegfr- ative,,,,, /N VEGFR- g lysate per lane. g lysate per well Logarithmically growing HEK cells expressing VEGFR- (negative) were treated with VEGF ( min; nm)(positive). Whole cell lysates were added to MSD MULTI-SOT -Spot plates coated with anti-vegfr- antibody on one of the four spatially distinct electrodes per well. hosphorylated VEGFR- was detected with anti-vegfr- antibody labeled with MSD SULFO-TAG reagent.

7 Detection of hosphorylated DGFR-, ositive, ative,,,, MSD MULTI-SOT -Well -Spot late A B anti-dgfr- SULFO-TAG antibody ositive/ative cell lysate anti-phospho-dgfr- antibody, ptyr os pdgfr- os pdgfr- ositive,,,,,, pdgfr- ative /N DGFR- g lysate per lane g lysate per well Serum-deprived NIHT cells (negative) were treated with DGF-BB ( ng/ml; min)(positive). Whole cell lysates were added to MSD MULTI-SOT -Spot plates coated with anti-phospho-dgfr- antibody on one of the four spatially distinct electrodes per well. hosphorylated DGFR- was detected with anti-dgfr- antibody labeled with MSD SULFO-TAG reagent.

8 Detection of hosphorylated and Total c-kit (ptyr) in the Same Well,,,,,,, ositive ative pc-kit tc-kit pc-kit tc-kit ,,,,,,,,,,,,,, pc-kit ositive,,,,,,, pc-kit ative /N MSD MULTI-SOT -Well -Spot late ptyr c-kit pc-kit c-kit os Total c-kit g lysate per lane SULFO-TAG detection antibody ositive/ative cell lysate Capture antibody. g lysate per well Growing MOe cells were starved in low serum for min (negative) and treated with SCF ( ng/ml; min)(positive). Whole cell lysates were added to MSD MULTI-SOT -Spot plates coated with anti-phospho-c-kit and anti-total-c-kit antibodies on two of the four spatially distinct electrodes per well. hosphorylated and total c-kit were detected with anti-total-c-kit antibody labeled with MSD SULFO-TAG reagent. os

9 Detection of hosphorylated and Total ErbB in the Same Well, ositive ative,,,,, ptyr ErbB MSD MULTI-SOT -Well -Spot late Total ErbB anti-erbb SULFO-TAG antibody ositive/ative cell lysate Capture antibody perbb terbb os perbb os perbb terbb ,,,,,,,,,,,,,, perbb ositive,,,,,,,,,,,,,,,,,,,, perbb ative,,,,,,, /N ErbB g lysate per lane g lysate per well Serum deprived SK-OV cells were treated with orthovanadate ( mm; hr) followed by EGF stimulation ( ng/ml; min)(positive) or Compound ( M; hr) and AG ( M; hr)(negative). Whole cell lysates were added to MSD MULTI-SOT -Spot plates coated with anti-phospho-erbb antibody and anti-total-erbb antibody on two of the four spatially distinct electrodes per well. hosphorylated and total ErbB were detected with anti-total-erbb antibody labeled with MSD SULFO-TAG reagent.

10 Determining % hosphoprotein: hosphorylated and Total Assay in the Same Well

11 Conclusions