Panel results of Cell based ELISA Assessing Activation of Cdc42 and Rac1
Active GTP-bound Rac1 and Cdc42 bind the downstream effector p21 activated kinase (PAK). The extent of PAK binding is thus a routine measure for quantifying activated Rac and Cdc42. Dr. Surviladze with the UNM Center for Molecular Discovery has demonstrated that the assay can be used to score for small molecules that activate or inhibit Rac nucleotide binding in vitro and in vivo. For further more ..
Depositor Specified Assays
University of New Mexico Assay Overview:
Assay Support: NIH I RO3 MH081231-01
HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases
PI: Angela Wandinger-Ness, Ph.D.
Co-PI: Larry Sklar, Ph.D.
Assay Development: Zurab Surviladze, Ph.D.
Assay Implementation: Elsa Romero, Anna Waller
Chemistry: University of Kansas Specialized Chemistry Center
KU Specialized Chemistry Center PI: Jeff Aube, Ph.D.
KU SCC Project Manager: Jennifer E. Golden. Ph.D.
KU SCC Chemists on this project: Chad Schroeder, M.S., Denise Simpson, Ph.D., Julica Noeth, B.S.
Assay Background and Significance:
Active GTP-bound Rac1 and Cdc42 bind the downstream effector p21 activated kinase (PAK). The extent of PAK binding is thus a routine measure for quantifying activated Rac and Cdc42. Dr. Surviladze with the UNM Center for Molecular Discovery has demonstrated that the assay can be used to score for small molecules that activate or inhibit Rac nucleotide binding in vitro and in vivo. For further evaluation and dose dependence assays of compounds found to be active in cell based morphological assays we will use an ELISA based version of the assay developed by Cytoskeleton, Inc. (GLISA, Denver, CO).
In the ELISA format PAK coated on microtiter wells was reconstituted in cold water immediately prior to the assay. 3T3 cells cultured in serum free medium and treated +/- epidermal growth factor (10 ng/ml) and sample molecules. No growth factor lysate served as baseline negative control, + growth factor lysate served as measure of maximal response. Assays conducted on different days were normalized based on maximal response. Cell lysates were prepared, snap frozen and equal amounts of cellular protein tested for Cdc42 or Rac1 activity. Dose range was measured in duplicate in multiple trials with sample molecule concentration of 1 microM and 10 microM. Readout was measured by colorimetric assay. Assay was run with a negative control (no cell lyaste, buffer with all other assay components) and a positive control (1 ng GTP-bound GTPase, with all other assay components) to allow calculation of ng active GTPase in each lysate sample.
The measured raw absorbance data were normalized between the different experiments as follows:
a) Subtract GLISA buffer blank from all Abs. values
b) Calculate nanograms active GTPase based on standard run in each experiment that corresponds to 1 nanogram GTP-Rac or GTP-Cdc42
c) Calculate average value for plus growth factor samples for each experiment and normalize all nanogram GTPase values to this value.
PUBCHEM_SCORE is based on the magnitude the response signal was depressed due to the presense of 10 microM sample compound. Thus PUBCHEM_SCORE = 100 * (1 - normalized nanogram GTPase). Active compounds have PUBCHEM_SCORE greater than 15.
** Test Concentration. § Panel component ID.