GAB1 (Phospho-Tyr627)Colorimetric Cell-Based ELISA Kit (CBCAB01533)

Product Name:	GAB1 (Phospho-Tyr627)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01533
ELISA Type:	Cell-Based
Target:	Phospho-GAB1 (Tyr627)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The GAB1 (Phospho-Tyr627) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GAB1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated GAB1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on GAB1 phosphorylation.

Qualitative determination of GAB1 (Phospho-Tyr627) concentration is achieved by an indirect ELISA format. In essence, GAB1 (Phospho-Tyr627) is captured by GAB1 (Phospho-Tyr627)-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: 2549, UniProt ID: Q13480, OMIM: 604439, Unigene: Hs.618456/Hs.80720
Gene Symbol:	GAB1
Sub Type:	Phospho

UniProt Protein Function:	GAB1: Adapter protein that plays a role in intracellular signaling cascades triggered by activated receptor-type kinases. Plays a role in FGFR1 signaling. Probably involved in signaling by the epidermal growth factor receptor (EGFR) and the insulin receptor (INSR). Interacts with GRB2 and with other SH2-containing proteins. Interacts with phosphorylated LAT2. Interacts with PTPRJ. Identified in a complex containing FRS2, GRB2, GAB1, PIK3R1 and SOS1. Interacts (phosphorylated) with PTPN11. Interacts with HCK. Belongs to the GAB family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Adaptor/scaffold; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 4q31.21 Cellular Component: cytosol Molecular Function:protein binding; SH3/SH2 adaptor activity; signal transducer activity Biological Process: activation of JNK activity; cell proliferation; epidermal growth factor receptor signaling pathway; epidermis development; fibroblast growth factor receptor signaling pathway; heart development; innate immune response; insulin receptor signaling pathway; nerve growth factor receptor signaling pathway; phosphoinositide-mediated signaling; platelet-derived growth factor receptor signaling pathway; regulation of cell migration; response to oxidative stress
NCBI Summary:	The protein encoded by this gene is a member of the IRS1-like multisubstrate docking protein family. It is an important mediator of branching tubulogenesis and plays a central role in cellular growth response, transformation and apoptosis. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Aug 2008]
UniProt Code:	Q13480
NCBI GenInfo Identifier:	90180201
NCBI Gene ID:	2549
NCBI Accession:	Q13480.2
UniProt Secondary Accession:	Q13480,Q4W5G2, Q6P1W2, A8K152,
UniProt Related Accession:	Q13480
Molecular Weight:	80,005 Da
NCBI Full Name:	GRB2-associated-binding protein 1
NCBI Synonym Full Names:	GRB2 associated binding protein 1
NCBI Official Symbol:	GAB1
NCBI Protein Information:	GRB2-associated-binding protein 1
UniProt Protein Name:	GRB2-associated-binding protein 1
UniProt Synonym Protein Names:	GRB2-associated binder 1; Growth factor receptor bound protein 2-associated protein 1
Protein Family:	GPI transamidase component
UniProt Gene Name:	GAB1
UniProt Entry Name:	GAB1_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-GAB1 (Phospho-Tyr627) Antibody, Anti-GAB1 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.)