Osteopontin Colorimetric Cell-Based ELISA Kit (CBCAB00188)

Product Name:	Osteopontin Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00188
ELISA Type:	Cell-Based
Target:	Osteopontin
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of Osteopontin concentration is achieved by an indirect ELISA format. In essence, Osteopontin is captured by Osteopontin-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: 6696, UniProt ID: P10451, OMIM: 166490, Unigene: Hs.313
Gene Symbol:	SPP1
Sub Type:	None

UniProt Protein Function:	osteopontin: Binds tightly to hydroxyapatite. Appears to form an integral part of the mineralized matrix. Probably important to cell-matrix interaction. Belongs to the osteopontin family. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 4q22.1 Cellular Component: extracellular space; cell projection; apical part of cell; perinuclear region of cytoplasm; extracellular region Molecular Function:protein binding; extracellular matrix binding; cytokine activity Biological Process: osteoblast differentiation; extracellular matrix disassembly; neutrophil chemotaxis; extracellular matrix organization and biogenesis; response to vitamin D; biomineral formation; response to steroid hormone stimulus; negative regulation of collateral sprouting of intact axon in response to injury; positive regulation of bone resorption; decidualization; cell adhesion; inflammatory response; embryo implantation
NCBI Summary:	The protein encoded by this gene is involved in the attachment of osteoclasts to the mineralized bone matrix. The encoded protein is secreted and binds hydroxyapatite with high affinity. The osteoclast vitronectin receptor is found in the cell membrane and may be involved in the binding to this protein. This protein is also a cytokine that upregulates expression of interferon-gamma and interleukin-12. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2011]
UniProt Code:	P10451
NCBI GenInfo Identifier:	129260
NCBI Gene ID:	6696
NCBI Accession:	P10451.1
UniProt Secondary Accession:	P10451,Q15681, Q15682, Q15683, Q4W597, Q567T5, Q8NBK2 Q96IZ1, B2RDA1,
UniProt Related Accession:	P10451
Molecular Weight:	33,843 Da
NCBI Full Name:	Osteopontin
NCBI Synonym Full Names:	secreted phosphoprotein 1
NCBI Official Symbol:	SPP1
NCBI Official Synonym Symbols:	OPN; BNSP; BSPI; ETA-1
NCBI Protein Information:	osteopontin; uropontin; nephropontin; SPP1/CALPHA1 fusion; urinary stone protein; early T-lymphocyte activation 1; osteopontin/immunoglobulin alpha 1 heavy chain constant region fusion protein; secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1)
UniProt Protein Name:	Osteopontin
UniProt Synonym Protein Names:	Bone sialoprotein 1; Nephropontin; Secreted phosphoprotein 1; SPP-1; Urinary stone protein; Uropontin
Protein Family:	Osteopontin
UniProt Gene Name:	SPP1
UniProt Entry Name:	OSTP_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-Osteopontin 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.)