Bookmark and Share
BioAssay: AID 2375

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 ..
_
   
 Tested Compounds
 Tested Compounds
All(7)
 
 
Inconclusive(7)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Inconclusive(7)
 
 
 Related BioAssays
 Related BioAssays
AID: 2375
Data Source: NMMLSC (UNM_ELISA_Cdc42_Rac1)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-02-18
Hold-until Date: 2010-08-15

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
1772Project utilizing multiplex HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPasessummaryGTPase Project Summary report
Description:
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).
Panel Information
ELISA for activated Cdc42 and Rac1 - Cell based ELISA assessing for active Cdc42 and Rac1
PID§NameSubstancePanel TargetsDescription
ActiveInactive
1Cdc4234Cell Division Cycle 42 GTP binding protein
2Rac132GTPase-activating protein 1

§ Panel component ID.
Protocol
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.

Calculations:

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.
Comment
Abbreviations: microM for micromolar, ng for nanogram, ml for milliliter
Result Definitions
Show more
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Cdc42_OutcomeOutcome for Cdc421Outcome
2Cdc42_ScorePubchem Score for Cdc42 based on measured value at 10 microM test compound1Integer
3AveCdc42_1microM (1μM**)Average normalized measurement of active Cdc42 in the presence of 1 microM test compound1Float
4StdCdc42_1microM (1μM**)Standard deviation of normalized measurement of active Cdc42 in the presence of 1 microM test compound1Float
5AveCdc42_10microM (10μM**)Average normalized measurement of active Cdc42 in the presence of 10 microM test compound1Float
6StdCdc42_10microM (10μM**)Standard deviation of normalized measurement of active Cdc42 in the presence of 10 microM test compound1Float
7Rac1_OutcomeOutcome for Rac12Outcome
8Rac1_ScorePubchem Score for Rac1 based on measured value at 10 microM test compound2Integer
9AveRac1_1microM (1μM**)Average normalized measurement of active Rac1 in the presence of 1 microM test compound2Float
10StdRac1_1microM (1μM**)Standard deviation of normalized measurement of active Rac1 in the presence of 1 microM test compound2Float
11AveRac1_10microM (10μM**)Average normalized measurement of active Rac1 in the presence of 10 microM test compound2Float
12StdRac1_10microM (10μM**)Standard deviation of normalized measurement of active Rac1 in the presence of 10 microM test compound2Float

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: NIH I RO3 MH081231-01

PageFrom: