FGFR1 Oncogene Partner Colorimetric Cell-Based ELISA Kit (CBCAB00656)

Product Name:	FGFR1 Oncogene Partner Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00656
ELISA Type:	Cell-Based
Target:	FGFR1 Oncogene Partner
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The FGFR1 Oncogene Partner Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect FGFR1 Oncogene Partner protein expression profile in cells. The kit can be used for measuring the relative amounts of FGFR1 Oncogene Partner in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on FGFR1 Oncogene Partner.

Qualitative determination of FGFR1 Oncogene Partner concentration is achieved by an indirect ELISA format. In essence, FGFR1 Oncogene Partner is captured by FGFR1 Oncogene Partner-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: 11116, UniProt ID: O95684, OMIM: 605392, Unigene: Hs.487175
Gene Symbol:	FGFR1OP
Sub Type:	None

UniProt Protein Function:	FOP: Required for anchoring microtubules to the centrosomes. A chromosomal aberration involving FGFR1OP may be a cause of stem cell myeloproliferative disorder (MPD). Translocation t(6;8)(q27;p11) with FGFR1. MPD is characterized by myeloid hyperplasia, eosinophilia and T-cell or B-cell lymphoblastic lymphoma. In general it progresses to acute myeloid leukemia. The fusion proteins FGFR1OP-FGFR1 or FGFR1-FGFR1OP may exhibit constitutive kinase activity and be responsible for the transforming activity. Belongs to the FGFR1OP family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Unknown function Chromosomal Location of Human Ortholog: 6q27 Cellular Component: centrosome; cytosol; nucleus; perinuclear region of cytoplasm Molecular Function:protein binding; protein homodimerization activity; protein kinase binding; protein tyrosine kinase inhibitor activity; protein-tyrosine kinase activity Biological Process: G2/M transition of mitotic cell cycle; negative regulation of protein kinase activity; positive regulation of cell growth; positive regulation of cell migration; positive regulation of cell proliferation
NCBI Summary:	This gene encodes a largely hydrophilic centrosomal protein that is required for anchoring microtubules to subcellular structures. A t(6;8)(q27;p11) chromosomal translocation, fusing this gene and the fibroblast growth factor receptor 1 (FGFR1) gene, has been found in cases of myeloproliferative disorder. The resulting chimeric protein contains the N-terminal leucine-rich region of this encoded protein fused to the catalytic domain of FGFR1. Alterations in this gene may also be associated with Crohn's disease, Graves' disease, and vitiligo. Alternatively spliced transcript variants that encode different proteins have been identified. [provided by RefSeq, Jul 2013]
UniProt Code:	O95684
NCBI GenInfo Identifier:	74762130
NCBI Gene ID:	11116
NCBI Accession:	O95684.1
UniProt Secondary Accession:	O95684,Q49AI0, Q5R3F6, Q96EW1, A8K1D1, B2R705,
UniProt Related Accession:	O95684
Molecular Weight:	16,106 Da
NCBI Full Name:	FGFR1 oncogene partner
NCBI Synonym Full Names:	FGFR1 oncogene partner
NCBI Official Symbol:	FGFR1OP
NCBI Official Synonym Symbols:	FOP
NCBI Protein Information:	FGFR1 oncogene partner
UniProt Protein Name:	FGFR1 oncogene partner
Protein Family:	FGFR1 oncogene partner
UniProt Gene Name:	FGFR1OP
UniProt Entry Name:	FR1OP_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-FGFR1 Oncogene Partner 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.)