GTPase Activating Protein (Phospho-Ser387)Colorimetric Cell-Based ELISA Kit (CBCAB01447)

Product Name:	GTPase Activating Protein (Phospho-Ser387)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01447
ELISA Type:	Cell-Based
Target:	GTPase Activating Protein (Phospho-Ser387)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The GTPase Activating Protein (Phospho-Ser387) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GTPase Activating Protein protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated GTPase Activating Protein in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on GTPase Activating Protein phosphorylation.

Qualitative determination of GTPase Activating Protein (Phospho-Ser387) concentration is achieved by an indirect ELISA format. In essence, GTPase Activating Protein (Phospho-Ser387) is captured by GTPase Activating Protein (Phospho-Ser387)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 29127, UniProt ID: Q9H0H5, OMIM: 604980, Unigene: Hs.505469
Gene Symbol:	RACGAP1
Sub Type:	Phospho

UniProt Protein Function:	MgcRacGAP: a rac GTPase activating protein. Plays an important role in cytokinesis by regulating cortical movement through RhoA. Phosphorylation by Aurora B apparently induces GAP activity toward RhoA.
UniProt Protein Details:	Protein type:GAPs; GAPs, Rac/Rho Chromosomal Location of Human Ortholog: 12q13.12 Cellular Component: nucleoplasm; microtubule; extrinsic to internal side of plasma membrane; acrosome; spindle midzone; midbody; nucleus; cytosol; cleavage furrow Molecular Function:gamma-tubulin binding; protein binding; phosphatidylinositol-3,4,5-triphosphate binding; metal ion binding; microtubule binding; beta-tubulin binding; protein kinase binding; GTPase activator activity; alpha-tubulin binding Biological Process: positive regulation of cytokinesis; regulation of attachment of spindle microtubules to kinetochore; spindle midzone assembly involved in mitosis; antigen processing and presentation of exogenous peptide antigen via MHC class II; sulfate transport; microtubule-based movement; regulation of small GTPase mediated signal transduction; embryonic development; cytokinesis after mitosis; small GTPase mediated signal transduction; neuroblast proliferation; spermatogenesis; blood coagulation; cytokinesis, contractile ring formation; positive regulation of GTPase activity
NCBI Summary:	The protein encoded by this gene belongs to the GTPase-activating protein (GAP) family. GAPs bind activated forms of Rho GTPases and stimulate GTP hydrolysis. Through this catalytic function, GAPs negatively regulate Rho-mediated signals. This protein plays a regulatory role in initiation of cytokinesis, controlling cell growth and differentiation of hematopoietic cells, regulating spermatogenesis, and in neuronal proliferation. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Sep 2011]
UniProt Code:	Q9H0H5
NCBI GenInfo Identifier:	74762727
NCBI Gene ID:	29127
NCBI Accession:	Q9H0H5.1
UniProt Secondary Accession:	Q9H0H5,Q6PJ26, Q9NWN2, Q9P250, Q9P2W2,
UniProt Related Accession:	Q9H0H5
Molecular Weight:	632
NCBI Full Name:	Rac GTPase-activating protein 1
NCBI Synonym Full Names:	Rac GTPase activating protein 1
NCBI Official Symbol:	RACGAP1
NCBI Official Synonym Symbols:	CYK4; ID-GAP; HsCYK-4; MgcRacGAP
NCBI Protein Information:	rac GTPase-activating protein 1; RACGAP1; protein CYK4 homolg; protein CYK4 homolog; male germ cell RacGap; GTPase activating protein
UniProt Protein Name:	Rac GTPase-activating protein 1
UniProt Synonym Protein Names:	Male germ cell RacGap; MgcRacGAP; Protein CYK4 homolog; CYK4; HsCYK-4
Protein Family:	Rac GTPase-activating protein
UniProt Gene Name:	RACGAP1
UniProt Entry Name:	RGAP1_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37 °C prior to adding cells.
2.	Incubate the cells for overnight at 37 °C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4 °C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-GTPase Activating Protein (Phospho-Ser387) Antibody, Anti-GTPase Activating Protein Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4 °C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)