Product Name:	Rat Apoptosis regulator BAX (Bax) ELISA Kit
SKU:	RTEB0892
Size:	96T
Target:	Rat Apoptosis regulator BAX (Bax)
Synonyms:	Apoptosis regulator BAX, Bax
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.312-20ng/mL
Sensitivity:	0.17ng/mL

Intra CV:

6.2%

Inter CV:

9.4%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	94-104%	104-112%	114-124%	109-117%
EDTA Plasma(N=5)	101-110%	92-101%	104-113%	98-108%
Heparin Plasma(N=5)	101-111%	97-107%	90-99%	87-97%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	109	103-115
Plasma	111	105-117

Function:

Accelerates programmed cell death by binding to, and antagonizing the apoptosis repressor BCL2 or its adenovirus homolog E1B 19k protein. Under stress conditions, undergoes a conformation change that causes translocation to the mitochondrion membrane, leading to the release of cytochrome c that then triggers apoptosis. Promotes activation of CASP3, and thereby apoptosis.

Uniprot:	Q63690
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Apoptosis regulator BAX
Sub Unit:	Homodimer. Forms higher oligomers under stress conditions. Interacts with BCL2L11. Interaction with BCL2L11 promotes BAX oligomerization and association with mitochondrial membranes, with subsequent release of cytochrome c. Forms heterodimers with BCL2, E1B 19K protein, BCL2L1 isoform Bcl-X(L), BCL2L2, MCL1 and A1. Interacts with SH3GLB1 and HN. Interacts with SFN and YWHAZ; the interaction occurs in the cytoplasm. Under stress conditions, JNK-mediated phosphorylation of SFN and YWHAZ, releases BAX to mitochondria. Interacts with RNF144B, which regulates the ubiquitin-dependent stability of BAX. Interacts with CLU under stress conditions that cause a conformation change leading to BAX oligomerization and association with mitochondria. Does not interact with CLU in unstressed cells (By similarity). Interacts with FAIM2/LFG2 (By similarity). Interacts with BOP (By similarity). Interacts (via a C-terminal 33 residues) with NOL3 (via CARD domain); inhibits BAX activation and translocationand consequently cytochrome c release from mitochondria.
Research Area:	Neurosciences
Subcellular Location:	Isoform Gamma Cytoplasm
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	BAX: Accelerates programmed cell death by binding to, and antagonizing the apoptosis repressor BCL2 or its adenovirus homolog E1B 19k protein. Under stress conditions, undergoes a conformation change that causes translocation to the mitochondrion membrane, leading to the release of cytochrome c that then triggers apoptosis. Promotes activation of CASP3, and thereby apoptosis. Homodimer. Forms higher oligomers under stress conditions. Interacts with BCL2L11. Interaction with BCL2L11 promotes BAX oligomerization and association with mitochondrial membranes, with subsequent release of cytochrome c. Forms heterodimers with BCL2, E1B 19K protein, BCL2L1 isoform Bcl-X(L), BCL2L2, MCL1 and A1. Interacts with SH3GLB1 and HN. Interacts with SFN and YWHAZ; the interaction occurs in the cytoplasm. Under stress conditions, JNK-mediated phosphorylation of SFN and YWHAZ, releases BAX to mitochondria. Isoform Sigma interacts with BCL2A1 and BCL2L1 isoform Bcl-X(L). Interacts with RNF144B, which regulates the ubiquitin-dependent stability of BAX. Interacts with CLU under stress conditions that cause a conformation change leading to BAX oligomerization and association with mitochondria. Does not interact with CLU in unstressed cells. Interacts with FAIM2/LFG2. Interacts with human cytomegalovirus/HHV-5 protein vMIA/UL37. Expressed in a wide variety of tissues. Isoform Psi is found in glial tumors. Isoform Alpha is expressed in spleen, breast, ovary, testis, colon and brain, and at low levels in skin and lung. Isoform Sigma is expressed in spleen, breast, ovary, testis, lung, colon, brain and at low levels in skin. Isoform Alpha and isoform Sigma are expressed in pro- myelocytic leukemia, histiocytic lymphoma, Burkitt's lymphoma, T- cell lymphoma, lymphoblastic leukemia, breast adenocarcinoma, ovary adenocarcinoma, prostate carcinoma, prostate adenocarcinoma, lung carcinoma, epidermoid carcinoma, small cell lung carcinoma and colon adenocarcinoma cell lines. Belongs to the Bcl-2 family. 8 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Tumor suppressor; Membrane protein, integral; Apoptosis; Mitochondrial Cellular Component: cytoplasm; cytosol; endoplasmic reticulum; endoplasmic reticulum membrane; integral to membrane; intracellular; membrane; mitochondrial membrane; mitochondrial outer membrane; mitochondrial permeability transition pore complex; mitochondrion; nuclear envelope; nucleus; perinuclear region of cytoplasm; pore complex Molecular Function:BH domain binding; BH3 domain binding; channel activity; chaperone binding; heat shock protein binding; identical protein binding; lipid binding; protein binding; protein complex binding; protein heterodimerization activity; protein homodimerization activity Biological Process: aging; apoptosis; apoptotic mitochondrial changes; B cell apoptosis; B cell homeostasis; B cell homeostatic proliferation; B cell negative selection; blood vessel remodeling; brain development; caspase activation; caspase activation via cytochrome c; cell proliferation; cellular respiration; cerebral cortex development; development of secondary sexual characteristics; DNA damage response, signal transduction resulting in induction of apoptosis; establishment and/or maintenance of transmembrane electrochemical gradient; fertilization; germ cell development; germ cell programmed cell death; glycosphingolipid metabolic process; homeostasis of number of cells; homeostasis of number of cells within a tissue; hypothalamus development; induction of apoptosis via death domain receptors; inner mitochondrial membrane organization and biogenesis; kidney development; leukocyte homeostasis; limb morphogenesis; male gonad development; mitochondrial fragmentation during apoptosis; mitochondrial fusion; myeloid cell homeostasis; negative regulation of cell proliferation; negative regulation of fibroblast proliferation; negative regulation of neuron apoptosis; negative regulation of peptidyl-serine phosphorylation; negative regulation of protein binding; nervous system development; neuron apoptosis; neuron migration; odontogenesis of dentine-containing teeth; outer mitochondrial membrane organization and biogenesis; ovarian follicle development; positive regulation of apoptosis; positive regulation of apoptosis involved in mammary gland involution; positive regulation of B cell apoptosis; positive regulation of neuron apoptosis; positive regulation of pigmentation; positive regulation of protein oligomerization; positive regulation of release of sequestered calcium ion into cytosol; post-embryonic camera-type eye morphogenesis; post-embryonic development; protein homooligomerization; protein insertion into mitochondrial membrane during induction of apoptosis; protein oligomerization; reduction of endoplasmic reticulum calcium ion concentration; regulation of caspase activity; regulation of cell cycle; regulation of mammary gland epithelial cell proliferation; regulation of mitochondrial membrane potential; regulation of neuron apoptosis; regulation of nitrogen utilization; regulation of protein heterodimerization activity; regulation of protein homodimerization activity; release of cytochrome c from mitochondria; release of matrix enzymes from mitochondria; response to axon injury; response to cocaine; response to copper ion; response to corticosterone stimulus; response to DNA damage stimulus; response to drug; response to gamma radiation; response to ionizing radiation; response to salt stress; response to toxin; response to wounding; retina development in camera-type eye; retinal cell programmed cell death; Sertoli cell proliferation; sex differentiation; spermatid differentiation; spermatogenesis; T cell homeostatic proliferation; transformed cell apoptosis; vagina development
NCBI Summary:	Bcl2-related gene; involved in the regulation of apoptotic cell death [RGD, Feb 2006]
UniProt Code:	Q63690
NCBI GenInfo Identifier:	2493273
NCBI Gene ID:	24887
NCBI Accession:	Q63690.2
UniProt Secondary Accession:	Q63690,Q62995, Q64383,
UniProt Related Accession:	Q63690
Molecular Weight:	Da
NCBI Full Name:	Apoptosis regulator BAX
NCBI Synonym Full Names:	Bcl2-associated X protein
NCBI Official Symbol:	Bax
NCBI Protein Information:	apoptosis regulator BAX
UniProt Protein Name:	Apoptosis regulator BAX
Protein Family:	Apoptosis regulator
UniProt Gene Name:	Bax
UniProt Entry Name:	BAX_RAT

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.