인간 TNFAIP1 (Tumor necrosis factor, alpha-induced protein 1, endothelial) ELISA

Product Name:	Human TNFAIP1 (Tumor necrosis factor, alpha-induced protein 1, endothelial) ELISA Kit
Product Code:	HUFI08365
Size:	96 Assays
Alias:	BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein 2 ELISA Kit, hBACURD2 ELISA Kit, BTB/POZ domain-containing protein TNFAIP1 ELISA Kit, Protein B12 ELISA Kit, Tumor necrosis factor ELISA Kit, alpha-induced protein 1 ELISA Kit, endothelial ELISA Kit, TNFAIP1 ELISA Kit, BACURD2 ELISA Kit, EDP1 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human TNFAIP1 (Tumor necrosis factor, alpha-induced protein 1, endothelial) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human TNFAIP1 (Tumor necrosis factor, alpha-induced protein 1, endothelial) and the recovery rates were calculated by comparing the measured value to the expected amount of Human TNFAIP1 (Tumor necrosis factor, alpha-induced protein 1, endothelial) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-102	95
EDTA plasma(n=5)	85-102	95
heparin plasma(n=5)	86-95	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human TNFAIP1 (Tumor necrosis factor, alpha-induced protein 1, endothelial) 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)	90-103%	87-103%	85-105%
EDTA plasma(n=5)	87-99%	90-97%	90-96%
UFH plasma(n=5)	85-97%	80-91%	92-98%

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 Protein Function:	TNFAIP1: Substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex involved in regulation of cytoskeleton structure. The BCR(BACURD2) E3 ubiquitin ligase complex mediates the ubiquitination of RHOA, leading to its degradation by the proteasome, thereby regulating the actin cytoskeleton and cell migration. Its interaction with RHOB may regulate apoptosis. May enhance the PCNA-dependent DNA polymerase delta activity. Belongs to the BACURD family.
UniProt Protein Details:	Protein type:DNA replication; Ubiquitin conjugating system Chromosomal Location of Human Ortholog: 17q22-q23 Cellular Component: cytoplasm; nucleolus; endosome Molecular Function:protein domain specific binding; protein binding; GTP-Rho binding; ubiquitin-protein ligase activity Biological Process: proteasomal ubiquitin-dependent protein catabolic process; embryonic development; cell migration; negative regulation of Rho protein signal transduction; apoptosis; stress fiber formation; protein ubiquitination; immune response; DNA replication; protein homooligomerization; positive regulation of DNA replication
NCBI Summary:	This gene was identified as a gene whose expression can be induced by the tumor necrosis factor alpha (TNF) in umbilical vein endothelial cells. Studies of a similar gene in mouse suggest that the expression of this gene is developmentally regulated in a tissue-specific manner. [provided by RefSeq, Jul 2008]
UniProt Code:	Q13829
NCBI GenInfo Identifier:	2833248
NCBI Gene ID:	7126
NCBI Accession:	Q13829.1
UniProt Secondary Accession:	Q13829,Q5TZQ1,
UniProt Related Accession:	Q13829
Molecular Weight:	36,204 Da
NCBI Full Name:	BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein 2
NCBI Synonym Full Names:	tumor necrosis factor, alpha-induced protein 1 (endothelial)
NCBI Official Symbol:	TNFAIP1
NCBI Official Synonym Symbols:	B12; B61; EDP1; BTBD34; hBACURD2
NCBI Protein Information:	BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein 2; BTB/POZ domain-containing protein TNFAIP1
UniProt Protein Name:	BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein 2
UniProt Synonym Protein Names:	BTB/POZ domain-containing protein TNFAIP1; Protein B12; Tumor necrosis factor, alpha-induced protein 1, endothelial
Protein Family:	BTB/POZ domain-containing adapter for CUL3-mediated RhoA degradation protein
UniProt Gene Name:	TNFAIP1
UniProt Entry Name:	BACD2_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.

Antibodies

Anti-TNFAIP1 Antibody (CAB17543)

TNFAIP1 Antibody (PACO01638)

TNFAIP1 Antibody (PACO12819)

TNFAIP1 Antibody (PACO15120)

TNFAIP1 Antibody (PACO15121)