Human S100A12 ELISA Kit (HUFI01441)- High Sensitivity

Product Name:	Human S100A12 ELISA Kit
Product Code:	HUFI01441
Size:	96 Assays
Alias:	S100A12, Protein S100-A12, S100 Calcium Binding Protein A12, S100 calcium-binding protein A12, Neutrophil S100 protein, MRP-6, Extracellular newly identified RAGE-binding protein, EN-RAGE, Calgranulin-C, CAGC, p6, CGRP, Calcium-binding protein in amniotic fluid 1, CAAF1, MRP6, CAAF1, MRP-6, ENRAGE
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human S100A12 concentrations in serum plasma and other biological fluids.
Sensitivity:	2.344pg/ml
Range:	3.906-250pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human S100A12 and the recovery rates were calculated by comparing the measured value to the expected amount of Human S100A12 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	90-101	96
EDTA plasma(n=5)	87-105	97
UFH plasma(n=5)	93-100	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human S100A12 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)	91-105%	86-104%	85-101%
EDTA plasma(n=5)	82-95%	83-95%	85-100%
UFH plasma(n=5)	81-100%	80-99%	82-96%

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

P80511

UniProt Protein Function:	S100A12: S100A12 is a calcium-, zinc- and copper-binding protein which plays a prominent role in the regulation of inflammatory processes and immune response. Its proinflammatory activity involves recruitment of leukocytes, promotion of cytokine and chemokine production, and regulation of leukocyte adhesion and migration. Acts as an alarmin or a danger associated molecular pattern (DAMP) molecule and stimulates innate immune cells via binding to receptor for advanced glycation endproducts (AGER). Binding to AGER activates the MAP-kinase and NF-kappa-B signaling pathways leading to production of proinflammatory cytokines and up-regulation of cell adhesion molecules ICAM1 and VCAM1. Acts as a monocyte and mast cell chemoattractant. Can stimulate mast cell degranulation and activation which generates chemokines, histamine and cytokines inducing further leukocyte recruitment to the sites of inflammation. Can inhibit the activity of matrix metalloproteinases; MMP2, MMP3 and MMP9 by chelating Zn(2+) from their active sites. Possesses filariacidal and filariastatic activity. Calcitermin possesses antifungal activity against C.albicans and is also active against E.coli and P.aeruginosa but not L.monocytogenes and S.aureus. Belongs to the S-101 family.
UniProt Protein Details:	Chromosomal Location of Human Ortholog: 1q21 Cellular Component: cytoskeleton; cytoplasm; extracellular region; plasma membrane; nucleus; cytosol Molecular Function:protein binding; RAGE receptor binding; copper ion binding; zinc ion binding; calcium ion binding Biological Process: neutrophil chemotaxis; positive regulation of MAP kinase activity; monocyte chemotaxis; positive regulation of I-kappaB kinase/NF-kappaB cascade; killing of cells of another organism; xenobiotic metabolic process; defense response to bacterium; innate immune response; inflammatory response; defense response to fungus; mast cell activation; cytokine secretion; activation of NF-kappaB transcription factor; positive regulation of inflammatory response
NCBI Summary:	The protein encoded by this gene is a member of the S100 family of proteins containing 2 EF-hand calcium-binding motifs. S100 proteins are localized in the cytoplasm and/or nucleus of a wide range of cells, and involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. S100 genes include at least 13 members which are located as a cluster on chromosome 1q21. This protein is proposed to be involved in specific calcium-dependent signal transduction pathways and its regulatory effect on cytoskeletal components may modulate various neutrophil activities. The protein includes an antimicrobial peptide which has antibacterial activity. [provided by RefSeq, Nov 2014]
UniProt Code:	P80511
NCBI GenInfo Identifier:	2507565
NCBI Gene ID:	6283
NCBI Accession:	P80511.2
UniProt Related Accession:	P80511
Molecular Weight:
NCBI Full Name:	Protein S100-A12
NCBI Synonym Full Names:	S100 calcium binding protein A12
NCBI Official Symbol:	S100A12Â Â
NCBI Official Synonym Symbols:	p6; CAGC; CGRP; MRP6; CAAF1; MRP-6; ENRAGEÂ Â
NCBI Protein Information:	protein S100-A12
UniProt Protein Name:	Protein S100-A12
UniProt Synonym Protein Names:	CGRP; Calcium-binding protein in amniotic fluid 1; CAAF1; Calgranulin-C; CAGC; Extracellular newly identified RAGE-binding protein; EN-RAGE; Migration inhibitory factor-related protein 6; MRP-6; p6; Neutrophil S100 protein; S100 calcium-binding protein A12Calcitermin
UniProt Gene Name:	S100A12Â Â
UniProt Entry Name:	S10AC_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 Âµl 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 Âµl 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.

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Human S100A12 ELISA Kit (HUFI01441)

Description