Product Name:	Human Bcl-2 ELISA Kit
Product Code:	HUFI00475
Size:	96 Assays
Alias:	Bcl-2, apoptosis regulator Bcl-2, B-cell CLL, lymphoma 2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human Bcl-2 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.938ng/ml
Range:	1.563-100ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human Bcl-2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human Bcl-2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-102	96
EDTA plasma(n=5)	85-103	93
UFH plasma(n=5)	87-100	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human Bcl-2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-98%	90-103%	88-101%
EDTA plasma(n=5)	82-101%	86-96%	83-97%
UFH plasma(n=5)	83-100%	85-100%	85-97%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P10415

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

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.