C-RAF (Phospho-Ser621)Colorimetric Cell-Based ELISA Kit (CBCAB01316)

Product Name:	C-RAF (Phospho-Ser621)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01316
ELISA Type:	Cell-Based
Target:	C-RAF (Phospho-Ser621)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The C-RAF (Phospho-Ser621) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect C-RAF protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated C-RAF in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on C-RAF phosphorylation.

Qualitative determination of C-RAF (Phospho-Ser621) concentration is achieved by an indirect ELISA format. In essence, C-RAF (Phospho-Ser621) is captured by C-RAF (Phospho-Ser621)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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: 5894, UniProt ID: P04049, OMIM: 164760/611553/611554, Unigene: Hs.159130
Gene Symbol:	RAF1
Sub Type:	Phospho

UniProt Protein Function:	RAF1: a proto-oncogenic TKL kinase of the RAF family. Functions downstream of the Ras family of membrane associated GTPases to which it binds directly. Once activated Raf-1 can phosphorylate and activate MEK1/2, which in turn phosphorylate and activate ERK1/2. Acts as a regulatory link between the membrane-associated Ras GTPases and the MAPK/ERK cascade, and this critical regulatory link functions as a switch determining cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation. RAF1 activation initiates a mitogen-activated protein kinase (MAPK) cascade that comprises a sequential phosphorylation of the dual-specific MAPK kinases (MEK1/2) and the extracellular signal-regulated kinases (ERK1/2). RAF1, subsequent to phosphorylation by PAK1, phosphorylates BAD (Bcl2-antagonist of cell death) at S75. Phosphorylates adenylyl cyclases ADCY2, ADCY5 and ADCY6, resulting in their activation. Phosphorylates PPP1R12A resulting in inhibition of the phosphatase activity. Phosphorylates TNNT2 (cardiac muscle troponin T). Can promote NF-kB activation and inhibit signal transducers involved in motility (ROCK2), apoptosis (ASK1 and MST2), proliferation and angiogenesis (RB1). Can protect cells from apoptosis also by translocating to the mitochondria where it binds BCL2 and displaces BAD. Restricts caspase activation in response to selected stimuli, notably Fas stimulation, pathogen-mediated macrophage apoptosis, and erythroid differentiation.
UniProt Protein Details:	Protein type:EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Oncoprotein; Kinase, protein; Protein kinase, TKL; TKL group; RAF family Chromosomal Location of Human Ortholog: 3p25 Cellular Component: Golgi apparatus; mitochondrial outer membrane; cytoplasm; plasma membrane; pseudopodium; nucleus; cytosol Molecular Function:small GTPase binding; protein serine/threonine kinase activity; identical protein binding; protein binding; MAP kinase kinase kinase activity; mitogen-activated protein kinase kinase binding; protein heterodimerization activity; metal ion binding; ATP binding; protein kinase activity Biological Process: axon guidance; nerve growth factor receptor signaling pathway; activation of MAPKK activity; wound healing; apoptosis; somatic stem cell maintenance; heart development; intermediate filament cytoskeleton organization and biogenesis; negative regulation of caspase activity; signal transduction; protein amino acid phosphorylation; regulation of apoptosis; negative regulation of cell proliferation; synaptic transmission; small GTPase mediated signal transduction; regulation of cell differentiation; transmembrane transport; epidermal growth factor receptor signaling pathway; platelet activation; fibroblast growth factor receptor signaling pathway; adenylate cyclase activation; MAPKKK cascade; positive regulation of peptidyl-serine phosphorylation; cell proliferation; Ras protein signal transduction; response to hypoxia; regulation of Rho protein signal transduction; insulin receptor signaling pathway; innate immune response; vascular endothelial growth factor receptor signaling pathway; blood coagulation; negative regulation of protein complex assembly; negative regulation of apoptosis Disease: Cardiomyopathy, Dilated, 1nn; Noonan Syndrome 5; Leopard Syndrome 2
NCBI Summary:	This gene is the cellular homolog of viral raf gene (v-raf). The encoded protein is a MAP kinase kinase kinase (MAP3K), which functions downstream of the Ras family of membrane associated GTPases to which it binds directly. Once activated, the cellular RAF1 protein can phosphorylate to activate the dual specificity protein kinases MEK1 and MEK2, which in turn phosphorylate to activate the serine/threonine specific protein kinases, ERK1 and ERK2. Activated ERKs are pleiotropic effectors of cell physiology and play an important role in the control of gene expression involved in the cell division cycle, apoptosis, cell differentiation and cell migration. Mutations in this gene are associated with Noonan syndrome 5 and LEOPARD syndrome 2. [provided by RefSeq, Jul 2008]
UniProt Code:	P04049
NCBI GenInfo Identifier:	125651
NCBI Gene ID:	5894
NCBI Accession:	P04049.1
UniProt Secondary Accession:	P04049,Q15278, Q9UC20, B0LPH8, B2R5N3,
UniProt Related Accession:	P04049
Molecular Weight:	648
NCBI Full Name:	RAF proto-oncogene serine/threonine-protein kinase
NCBI Synonym Full Names:	Raf-1 proto-oncogene, serine/threonine kinase
NCBI Official Symbol:	RAF1
NCBI Official Synonym Symbols:	NS5; CRAF; Raf-1; c-Raf; CMD1NN
NCBI Protein Information:	RAF proto-oncogene serine/threonine-protein kinase; Oncogene RAF1; proto-oncogene c-RAF; C-Raf proto-oncogene, serine/threonine kinase; v-raf-1 murine leukemia viral oncogene homolog 1; raf proto-oncogene serine/threonine protein kinase
UniProt Protein Name:	RAF proto-oncogene serine/threonine-protein kinase
UniProt Synonym Protein Names:	Proto-oncogene c-RAF; cRaf; Raf-1
Protein Family:	Rik1-associated factor
UniProt Gene Name:	RAF1
UniProt Entry Name:	RAF1_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-C-RAF (Phospho-Ser621) Antibody, Anti-C-RAF 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.)