BCL-2 (Phospho-Ser70)Colorimetric Cell-Based ELISA Kit (CBCAB01501)

Product Name:	BCL-2 (Phospho-Ser70)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01501
ELISA Type:	Cell-Based
Target:	BCL-2 (Phospho-Ser70)
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The BCL-2 (Phospho-Ser70) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect BCL-2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated BCL-2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on BCL-2 phosphorylation.

Qualitative determination of BCL-2 (Phospho-Ser70) concentration is achieved by an indirect ELISA format. In essence, BCL-2 (Phospho-Ser70) is captured by BCL-2 (Phospho-Ser70)-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: 596, UniProt ID: P10415, OMIM: 151430, Unigene: Hs.150749
Gene Symbol:	BCL2
Sub Type:	Phospho

UniProt Protein Function:	Bcl-2: a antiapoptotic member of the Bcl-2 family. Regulates cell death by controlling the mitochondrial membrane permeability. Inhibits caspase activity either by preventing the release of cytochrome c from the mitochondria and/or by binding to the apoptosis-activating factor (APAF-1). Phosphorylation by JNKs may increase its antiapoptotic functions.
UniProt Protein Details:	Protein type:Autophagy; Membrane protein, integral; Oncoprotein; Apoptosis Chromosomal Location of Human Ortholog: 18q21.3 Cellular Component: pore complex; endoplasmic reticulum membrane; mitochondrial outer membrane; nuclear membrane; mitochondrion; membrane; endoplasmic reticulum; cytoplasm; nucleus; cytosol; myelin sheath Molecular Function:identical protein binding; protein binding; protein homodimerization activity; protease binding; protein phosphatase 2A binding; protein heterodimerization activity; channel activity; sequence-specific DNA binding; ubiquitin protein ligase binding; BH3 domain binding; channel inhibitor activity; transcription factor binding Biological Process: response to nicotine; focal adhesion formation; positive regulation of catalytic activity; developmental growth; renal system process; protein polyubiquitination; pigment granule organization and biogenesis; response to toxin; response to glucocorticoid stimulus; T cell differentiation in the thymus; ear development; lymphoid progenitor cell differentiation; positive regulation of multicellular organism growth; female pregnancy; glomerulus development; negative regulation of mitochondrial depolarization; post-embryonic development; cochlear nucleus development; cellular response to glucose starvation; negative regulation of myeloid cell apoptosis; B cell receptor signaling pathway; regulation of mitochondrial membrane potential; positive regulation of B cell proliferation; negative regulation of ossification; regulation of transmembrane transporter activity; T cell homeostasis; negative regulation of neuron apoptosis; cell growth; defense response to virus; spleen development; response to drug; positive regulation of neuron maturation; release of cytochrome c from mitochondria; regulation of protein homodimerization activity; axon regeneration; actin filament organization; cell aging; digestive tract morphogenesis; regulation of calcium ion transport; positive regulation of cell growth; organ growth; DNA damage response, signal transduction resulting in induction of apoptosis; induction of apoptosis via death domain receptors; gland morphogenesis; negative regulation of osteoblast proliferation; regulation of mitochondrial membrane permeability; regulation of nitrogen utilization; metanephros development; oocyte development; negative regulation of apoptosis; B cell proliferation; negative regulation of autophagy; regulation of protein heterodimerization activity; behavioral fear response; melanin metabolic process; regulation of cell-matrix adhesion; apoptosis; negative regulation of retinal cell programmed cell death; regulation of protein stability; positive regulation of smooth muscle cell migration; protein amino acid dephosphorylation; response to radiation; ovarian follicle development; positive regulation of skeletal muscle fiber development; B cell homeostasis; positive regulation of melanocyte differentiation; melanocyte differentiation; response to gamma radiation; negative regulation of cellular pH reduction; transmembrane transport; response to iron ion; regulation of viral genome replication; negative regulation of cell migration; mesenchymal cell development; ossification; hair follicle morphogenesis; CD8-positive, alpha-beta T cell lineage commitment; thymus development; B cell lineage commitment; male gonad development; peptidyl-threonine phosphorylation; positive regulation of peptidyl-serine phosphorylation; humoral immune response; response to UV-B; endoplasmic reticulum calcium ion homeostasis; neuron apoptosis; peptidyl-serine phosphorylation; response to hydrogen peroxide; axonogenesis; ureteric bud branching; homeostasis of number of cells within a tissue; response to cytokine stimulus; innate immune response; negative regulation of cell growth; induction of apoptosis by oxidative stress; response to DNA damage stimulus
NCBI Summary:	This gene encodes an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells such as lymphocytes. Constitutive expression of BCL2, such as in the case of translocation of BCL2 to Ig heavy chain locus, is thought to be the cause of follicular lymphoma. Two transcript variants, produced by alternate splicing, differ in their C-terminal ends. [provided by RefSeq, Jul 2008]
UniProt Code:	P10415
NCBI GenInfo Identifier:	231632
NCBI Gene ID:	596
NCBI Accession:	P10415.2
UniProt Secondary Accession:	P10415,P10416, Q13842, Q16197, C9JHD5,
UniProt Related Accession:	P10415
Molecular Weight:	239
NCBI Full Name:	Apoptosis regulator Bcl-2
NCBI Synonym Full Names:	B-cell CLL/lymphoma 2
NCBI Official Symbol:	BCL2
NCBI Official Synonym Symbols:	Bcl-2; PPP1R50
NCBI Protein Information:	apoptosis regulator Bcl-2; protein phosphatase 1, regulatory subunit 50
UniProt Protein Name:	Apoptosis regulator Bcl-2
Protein Family:	Bcl2-associated agonist of cell death
UniProt Gene Name:	BCL2
UniProt Entry Name:	BCL2_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-BCL-2 (Phospho-Ser70) Antibody, Anti-BCL-2 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.)