Retinoblastoma (Phospho-Ser807) Cell-Based ELISA Kit

Product Name:	Retinoblastoma (Phospho-Ser807)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00483
ELISA Type:	Cell-Based
Target:	Retinoblastoma (Phospho-Ser807)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Retinoblastoma (Phospho-Ser807) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Retinoblastoma protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Retinoblastoma in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Retinoblastoma phosphorylation.

Qualitative determination of Retinoblastoma (Phospho-Ser807) concentration is achieved by an indirect ELISA format. In essence, Retinoblastoma (Phospho-Ser807) is captured by Retinoblastoma (Phospho-Ser807)-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: 5925, UniProt ID: P06400, OMIM: 109800/180200/259500, Unigene: Hs.408528
Gene Symbol:	RB1
Sub Type:	Phospho

UniProt Protein Function:	Rb: retinoblastoma tumor suppressor protein regulates cell proliferation by controlling progression through the G1-phase restriction point. Has three distinct binding domains and interacts with regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase and proteins with a conserved LXCXE motif. Cell cycle-dependent phosphorylation by Cdks inhibits Rb target binding, thus allowing cell cycle progression. Recruits and targets histone methyltransferase suv39h1 leading to epigenetic transcriptional repression.
UniProt Protein Details:	Protein type:Oncoprotein; Transcription, coactivator/corepressor; Nuclear receptor co-regulator; Tumor suppressor Chromosomal Location of Human Ortholog: 13q14.2 Cellular Component: nucleoplasm; SWI/SNF complex; PML body; Rb-E2F complex; spindle; chromatin; nucleus Molecular Function:identical protein binding; protein binding; androgen receptor binding; DNA binding; ubiquitin protein ligase binding; transcription coactivator activity; phosphoprotein binding; transcription factor binding; kinase binding; transcription factor activity Biological Process: sister chromatid biorientation; viral reproduction; positive regulation of transcription, DNA-dependent; negative regulation of transcription from RNA polymerase II promoter, mitotic; regulation of mitotic cell cycle; neuron maturation; positive regulation of macrophage differentiation; cell cycle arrest; neurite development; negative regulation of epithelial cell proliferation; transcription, DNA-dependent; negative regulation of transcription factor activity; mitotic cell cycle checkpoint; enucleate erythrocyte differentiation; regulation of lipid kinase activity; chromatin remodeling; G1/S-specific transcription in mitotic cell cycle; myoblast differentiation; neuron apoptosis; maintenance of mitotic sister chromatid cohesion; gut development; cell division; neuron morphogenesis during differentiation; negative regulation of smoothened signaling pathway; androgen receptor signaling pathway; Ras protein signal transduction; positive regulation of mitotic metaphase/anaphase transition; negative regulation of protein kinase activity; striated muscle cell differentiation; positive regulation of transcription from RNA polymerase II promoter; mitotic cell cycle; negative regulation of transcription, DNA-dependent; cell cycle checkpoint; G1/S transition of mitotic cell cycle Disease: Bladder Cancer; Osteogenic Sarcoma; Small Cell Cancer Of The Lung; Retinoblastoma
NCBI Summary:	The protein encoded by this gene is a negative regulator of the cell cycle and was the first tumor suppressor gene found. The encoded protein also stabilizes constitutive heterochromatin to maintain the overall chromatin structure. The active, hypophosphorylated form of the protein binds transcription factor E2F1. Defects in this gene are a cause of childhood cancer retinoblastoma (RB), bladder cancer, and osteogenic sarcoma. [provided by RefSeq, Jul 2008]
UniProt Code:	P06400
NCBI GenInfo Identifier:	132164
NCBI Gene ID:	5925
NCBI Accession:	P06400.2
UniProt Secondary Accession:	P06400,P78499, Q5VW46, Q8IZL4, A8K5E3,
UniProt Related Accession:	P06400
Molecular Weight:	928
NCBI Full Name:	Retinoblastoma-associated protein
NCBI Synonym Full Names:	retinoblastoma 1
NCBI Official Symbol:	RB1
NCBI Official Synonym Symbols:	RB; pRb; OSRC; pp110; p105-Rb; PPP1R130
NCBI Protein Information:	retinoblastoma-associated protein; retinoblastoma suspectibility protein; prepro-retinoblastoma-associated protein; protein phosphatase 1, regulatory subunit 130
UniProt Protein Name:	Retinoblastoma-associated protein
UniProt Synonym Protein Names:	p105-Rb; pRb; Rb; pp110
Protein Family:	Retinoblastoma-associated protein
UniProt Gene Name:	RB1
UniProt Entry Name:	RB_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-Retinoblastoma (Phospho-Ser807) Antibody, Anti-Retinoblastoma 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.)