GIT2 Colorimetric Cell-Based ELISA

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SKU:
CBCAB01094
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Reactivity:
Mouse
Reactivity:
Rat
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:GIT2 Colorimetric Cell-Based ELISA
Product Code:CBCAB01094
ELISA Type:Cell-Based
Target:GIT2
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The GIT2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GIT2 protein expression profile in cells. The kit can be used for measuring the relative amounts of GIT2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GIT2.

Qualitative determination of GIT2 concentration is achieved by an indirect ELISA format. In essence, GIT2 is captured by GIT2-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary 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: 9815, UniProt ID: Q14161, OMIM: 608564, Unigene: Hs.434996
Gene Symbol:GIT2
Sub Type:None
UniProt Protein Function:GIT2: possesses ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) activity. Interacts with G protein-coupled receptor kinases. Associates with paxillin. Also interacts with PIX exchange factors. Regulates Golgi and actin cytoskeletal organization. Important in integrin-mediated cell adhesion. Ten alternatively spliced isoforms have been described and additional isoforms seem to exist. Contains 1 Arf-GAP domain and 3 ANK repeats.
UniProt Protein Details:

Protein type:GAPs; GAPs, ARF; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 12q24.1

Cellular Component: focal adhesion; nucleoplasm

Molecular Function:protein binding

Biological Process: regulation of G-protein coupled receptor protein signaling pathway

NCBI Summary:This gene encodes a member of the GIT protein family, which interact with G protein-coupled receptor kinases and possess ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) activity. GIT proteins traffic between cytoplasmic complexes, focal adhesions, and the cell periphery, and interact with Pak interacting exchange factor beta (PIX) to form large oligomeric complexes that transiently recruit other proteins. GIT proteins regulate cytoskeletal dynamics and participate in receptor internalization and membrane trafficking. This gene has been shown to repress lamellipodial extension and focal adhesion turnover, and is thought to regulate cell motility. This gene undergoes extensive alternative splicing to generate multiple isoforms, but the full-length nature of some of these variants has not been determined. The various isoforms have functional differences, with respect to ARF GAP activity and to G protein-coupled receptor kinase 2 binding. [provided by RefSeq, Sep 2008]
UniProt Code:Q14161
NCBI GenInfo Identifier:17376322
NCBI Gene ID:9815
NCBI Accession:Q14161.2
UniProt Secondary Accession:Q14161,Q86U59, Q96CI2, Q9BV91, Q9Y5V2,
UniProt Related Accession:Q14161
Molecular Weight:70,183 Da
NCBI Full Name:ARF GTPase-activating protein GIT2
NCBI Synonym Full Names:GIT ArfGAP 2
NCBI Official Symbol:GIT2  
NCBI Official Synonym Symbols:PKL; CAT2; CAT-2  
NCBI Protein Information:ARF GTPase-activating protein GIT2
UniProt Protein Name:ARF GTPase-activating protein GIT2
UniProt Synonym Protein Names:Cool-interacting tyrosine-phosphorylated protein 2; CAT-2; CAT2; G protein-coupled receptor kinase-interactor 2; GRK-interacting protein 2
Protein Family:ARF GTPase-activating protein
UniProt Gene Name:GIT2  
UniProt Entry Name:GIT2_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-GIT2 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.)

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