Human cIAP-1 ELISA Kit (HUFI00959)- High Sensitivity

Product Name:	Human Baculoviral IAP repeat-containing protein 2/cIAP-1 ELISA Kit
Product Code:	HUFI00959
Size:	96 Assays
Alias:	BIRC2, cIAP?1, HIAP?2, MIHB, API1Hiap-2, apoptosis inhibitor 1, baculoviral IAP repeat-containing 2, baculoviral IAP repeat-containing protein 2, cIAP1, c-IAP1, hIAP2, IAP homolog B, IAP2, IAP-2, Inhibitor of apoptosis protein 2, NFR2-TRAF signalling complex protein
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human BIRC2 concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human BIRC2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human BIRC2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-100	94
EDTA plasma(n=5)	89-105	99
UFH plasma(n=5)	85-99	91

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human BIRC2 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%	94-104%	90-105%
EDTA plasma(n=5)	92-99%	84-96%	86-100%
UFH plasma(n=5)	83-95%	89-99%	82-99%

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

Q13490

UniProt Protein Function:	BIRC2: Multi-functional protein which regulates not only caspases and apoptosis, but also modulates inflammatory signaling and immunity, mitogenic kinase signaling, and cell proliferation, as well as cell invasion and metastasis. Acts as an E3 ubiquitin- protein ligase regulating NF-kappa-B signaling and regulates both canonical and non-canonical NF-kappa-B signaling by acting in opposite directions: acts as a positive regulator of the canonical pathway and suppresses constitutive activation of non-canonical NF-kappa-B signaling. The target proteins for its E3 ubiquitin- protein ligase activity include: RIPK1, RIPK2, RIPK3, RIPK4, CASP3, CASP7, CASP8, TRAF2, DIABLO/SMAC, MAP3K14/NIK, MAP3K5/ASK1, IKBKG/NEMO and MXD1/MAD1. Can also function as an E3 ubiquitin- protein ligase of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Acts as an important regulator of innate immune signaling via regulation of Toll-like receptors (TLRs), Nodlike receptors (NLRs) and RIG-I like receptors (RLRs), collectively referred to as pattern recognition receptors (PRRs). Protects cells from spontaneous formation of the ripoptosome, a large multi-protein complex that has the capability to kill cancer cells in a caspase-dependent and caspase-independent manner. Suppresses ripoptosome formation by ubiquitinating RIPK1 and CASP8. Can stimulate the transcriptional activity of E2F1. Plays a role in the modulation of the cell cycle. Interacts with DIABLO/SMAC and with PRSS25; these interactions inhibit apoptotic suppressor activity. Interacts with CASP9. Interacts (via BIR domains) with TRAF2. Interacts with E2F1, RIPK1, RIPK2, RIPK3, RIPK4, BIRC5/survivin and USP19. Present in many fetal and adult tissues. Mainly expressed in adult skeletal muscle, thymus, testis, ovary, and pancreas, low or absent in brain and peripheral blood leukocytes. The CARD domain inhibits the activation of E3 ubiquitin ligase activity by preventing RING domain dimerization and E2 ubiquitin donor binding and activation. The CARD domain- mediated autoinhibition of the E3 ubiquitin-protein ligase activity suppresses cell proliferation and migration. USP19 regulates the stability of BIRC2/c-IAP1 by preventing its ubiquitination. Belongs to the IAP family.
UniProt Protein Details:	Protein type:Ubiquitin ligase; EC 6.3.2.-; Ubiquitin conjugating system; Ligase Chromosomal Location of Human Ortholog: 11q22 Cellular Component: XY body; internal side of plasma membrane; spindle microtubule; cytoplasm; nucleus; cytosol; lipid raft Molecular Function:protein binding; zinc ion binding; caspase inhibitor activity; transcription coactivator activity; ubiquitin-protein ligase activity; protein N-terminus binding; ligase activity Biological Process: proteasomal ubiquitin-dependent protein catabolic process; regulation of toll-like receptor signaling pathway; positive regulation of I-kappaB kinase/NF-kappaB cascade; protein polyubiquitination; response to cAMP; transcription, DNA-dependent; apoptosis; regulation of cell cycle; protein heterooligomerization; MyD88-independent toll-like receptor signaling pathway; toll-like receptor 3 signaling pathway; regulation of innate immune response; regulation of cell proliferation; regulation of apoptosis; response to ethanol; cell surface receptor linked signal transduction; regulation of transcription, DNA-dependent; regulation of cell differentiation; regulation of inflammatory response; response to hypoxia; toll-like receptor signaling pathway; innate immune response; toll-like receptor 4 signaling pathway; placenta development; cell structure disassembly during apoptosis; negative regulation of apoptosis
NCBI Summary:	The protein encoded by this gene is a member of a family of proteins that inhibits apoptosis by binding to tumor necrosis factor receptor-associated factors TRAF1 and TRAF2, probably by interfering with activation of ICE-like proteases. This encoded protein inhibits apoptosis induced by serum deprivation and menadione, a potent inducer of free radicals. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jan 2012]
UniProt Code:	Q13490
NCBI GenInfo Identifier:	2497238
NCBI Gene ID:	329
NCBI Accession:	Q13490.2
UniProt Secondary Accession:	Q13490,Q16516, Q4TTG0, B4E026,
UniProt Related Accession:	Q13490
Molecular Weight:	618
NCBI Full Name:	Baculoviral IAP repeat-containing protein 2
NCBI Synonym Full Names:	baculoviral IAP repeat containing 2
NCBI Official Symbol:	BIRC2
NCBI Official Synonym Symbols:	API1; MIHB; HIAP2; RNF48; cIAP1; Hiap-2; c-IAP1
NCBI Protein Information:	baculoviral IAP repeat-containing protein 2; IAP-2; IAP homolog B; apoptosis inhibitor 1; RING finger protein 48; inhibitor of apoptosis protein 2; baculoviral IAP repeat-containing 2; NFR2-TRAF signalling complex protein; TNFR2-TRAF-signaling complex protein 2
UniProt Protein Name:	Baculoviral IAP repeat-containing protein 2
UniProt Synonym Protein Names:	C-IAP1; IAP homolog B; Inhibitor of apoptosis protein 2; IAP-2; hIAP-2; hIAP2; RING finger protein 48; TNFR2-TRAF-signaling complex protein 2
Protein Family:	Baculoviral IAP repeat-containing protein
UniProt Gene Name:	BIRC2
UniProt Entry Name:	BIRC2_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.