G3BP-1 (Phospho-Ser232)Colorimetric Cell-Based ELISA Kit (CBCAB01532)

Product Name:	G3BP-1 (Phospho-Ser232)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01532
ELISA Type:	Cell-Based
Target:	G3BP-1 (Phospho-Ser232)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The G3BP-1 (Phospho-Ser232) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect G3BP-1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated G3BP-1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on G3BP-1 phosphorylation.

Qualitative determination of G3BP-1 (Phospho-Ser232) concentration is achieved by an indirect ELISA format. In essence, G3BP-1 (Phospho-Ser232) is captured by G3BP-1 (Phospho-Ser232)-specific primary (1Ã¸) antibodies while the HRP-conjugated secondary (2Ã¸) antibodies bind the Fc region of the 1Ã¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ã¸ 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: 10146, UniProt ID: Q13283, OMIM: 608431, Unigene: Hs.587054
Gene Symbol:	G3BP1
Sub Type:	Phospho

UniProt Protein Function:	G3BP-1: an hnRNA-binding protein and endoribonuclease that participates in the Ras signal transduction pathway. A regulated effector of stress granule (SG) assembly. SGs are involved in mRNA sorting in the storing of untranslated mRNAs. Cleaves exclusively between cytosine and adenine and cleaves MYC mRNA preferentially at the 3'-UTR. ATP- and magnesium-dependent helicase. Unwinds preferentially partial DNA and RNA duplexes having a 17 bp annealed portion and either a hanging 3' tail or hanging tails at both 5'- and 3'-ends. Unwinds DNA/DNA, RNA/DNA, and RNA/RNA substrates with comparable efficiency. Acts unidirectionally by moving in the 5' to 3' direction along the bound single-stranded DNA. Binds to the SH3 domain of Ras GTPase-activating protein (RasGAP) in proliferating cells but not in quiescent cells. Cytoplasmic in proliferating cells, can be recruited to the plasma membrane in exponentially growing cells. Cytosolic and partially nuclear in resting cells. Recruited to SGs upon either arsenite or high temperature treatment. RasGAP-dependent phosphorylation of S149 induces a conformational change that prevents self-association. Dephosphorylation after HRAS activation is required for stress granule assembly. S149 phosphorylation induces partial nuclear localization. A component of a TAU mRNP complex, Interacts with USP10, and may regulate it.
UniProt Protein Details:	Protein type:RNA-binding; Helicase; Adaptor/scaffold; EC 3.6.4.12; EC 3.6.4.13 Chromosomal Location of Human Ortholog: 5q33.1 Cellular Component: focal adhesion; stress granule; cytoplasm; plasma membrane; nucleus; cytosol Molecular Function:mRNA binding; ATP-dependent DNA helicase activity; protein binding; DNA binding; endonuclease activity; ATP-dependent RNA helicase activity; ATP binding Biological Process: transport; metabolic process; Ras protein signal transduction; DNA duplex unwinding
NCBI Summary:	This gene encodes one of the DNA-unwinding enzymes which prefers partially unwound 3'-tailed substrates and can also unwind partial RNA/DNA and RNA/RNA duplexes in an ATP-dependent fashion. This enzyme is a member of the heterogeneous nuclear RNA-binding proteins and is also an element of the Ras signal transduction pathway. It binds specifically to the Ras-GTPase-activating protein by associating with its SH3 domain. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. [provided by RefSeq, Jul 2008]
UniProt Code:	Q13283
NCBI GenInfo Identifier:	14916572
NCBI Gene ID:	10146
NCBI Accession:	Q13283.1
UniProt Secondary Accession:	Q13283,Q5HYE9,
UniProt Related Accession:	Q13283
Molecular Weight:	466
NCBI Full Name:	Ras GTPase-activating protein-binding protein 1
NCBI Synonym Full Names:	GTPase activating protein (SH3 domain) binding protein 1
NCBI Official Symbol:	G3BP1
NCBI Official Synonym Symbols:	G3BP; HDH-VIII
NCBI Protein Information:	ras GTPase-activating protein-binding protein 1; G3BP-1; GAP binding protein; ATP-dependent DNA helicase VIII; GAP SH3 domain-binding protein 1; RasGAP-associated endoribonuclease G3BP; Ras-GTPase-activating protein SH3-domain-binding protein
UniProt Protein Name:	Ras GTPase-activating protein-binding protein 1
UniProt Synonym Protein Names:	ATP-dependent DNA helicase VIII; hDH VIII; GAP SH3 domain-binding protein 1
Protein Family:	Ras GTPase-activating protein-binding protein
UniProt Gene Name:	G3BP1
UniProt Entry Name:	G3BP1_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-G3BP-1 (Phospho-Ser232) Antibody, Anti-G3BP-1 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.)