Product Name:	Mouse S100A8 ELISA Kit
Product Code:	MOFI00488
Size:	96 Assays
Alias:	S100A8, Calgranulin A, CFAG, MRP8, 60B8AG, CAGACP-10, calgranulin A, calgranulin-A, Calgranulin-A, Calprotectin L1L subunit, CFAGL1Ag, CGLA, Cystic fibrosis antigen, Leukocyte L1 complex light chain, MA387, MIF, MigRation inhibitory factor-related protein 8, MRP-8, MRP8S100 calcium binding protein A8, calgranulin A, NIF, P8, p8, protein S100-A8, S100 calcium binding protein A8, S100 calcium-binding protein A8, S100 calcium-binding protein A8, calgranulin A, Urinary stone protein band A
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse S100A8 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.375ng/ml
Range:	0.625-40ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Mouse S100A8 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse S100A8 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-102	95
EDTA plasma(n=5)	92-104	98
UFH plasma(n=5)	85-102	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse S100A8 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)	94-104%	92-105%	87-104%
EDTA plasma(n=5)	84-97%	82-98%	87-99%
UFH plasma(n=5)	80-97%	87-93%	86-100%

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

P27005

UniProt Protein Function:	S100A8: S100A8 is a calcium- and zinc-binding protein which plays a prominent role in the regulation of inflammatory processes and immune response. It can induce neutrophil chemotaxis and adhesion. Predominantly found as calprotectin (S100A8/A9) which has a wide plethora of intra- and extracellular functions. The intracellular functions include: facilitating leukocyte arachidonic acid trafficking and metabolism, modulation of the tubulin-dependent cytoskeleton during migration of phagocytes and activation of the neutrophilic NADPH-oxidase. Activates NADPH- oxidase by facilitating the enzyme complex assembly at the cell membrane, transfering arachidonic acid, an essential cofactor, to the enzyme complex and S100A8 contributes to the enzyme assembly by directly binding to NCF2/P67PHOX. The extracellular functions involve proinfammatory, antimicrobial, oxidant-scavenging and apoptosis-inducing activities. Its proinflammatory activity includes 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 pattern recognition receptors such as Toll-like receptor 4 (TLR4) and receptor for advanced glycation endproducts (AGER). Binding to TLR4 and AGER activates the MAP-kinase and NF- kappa-B signaling pathways resulting in the amplification of the proinflammatory cascade. Has antimicrobial activity towards bacteria and fungi and exerts its antimicrobial activity probably via chelation of Zn(2+) which is essential for microbial growth. Can induce cell death via autophagy and apoptosis and this occurs through the cross-talk of mitochondria and lysosomes via reactive oxygen species (ROS) and the process involves BNIP3. Can regulate neutrophil number and apoptosis by an anti-apoptotic effect; regulates cell survival via ITGAM/ITGB and TLR4 and a signaling mechanism involving MEK-ERK. Its role as an oxidant scavenger has a protective role in preventing exaggerated tissue damage by scavenging oxidants. Can act as a potent amplifier of inflammation in autoimmunity as well as in cancer development and tumor spread. Belongs to the S-100 family.
UniProt Protein Details:	Chromosomal Location of Human Ortholog: 3 F1\|3 39.9 cM Cellular Component: extracellular space; nucleus Biological Process: astrocyte development; autophagy; caspase activation; leukocyte migration during inflammatory response; neutrophil chemotaxis; peptidyl-cysteine S-nitrosylation; positive regulation of inflammatory response; positive regulation of peptide secretion
UniProt Code:	P27005
NCBI GenInfo Identifier:	1173338
NCBI Gene ID:	20201
NCBI Accession:	P27005.3
UniProt Secondary Accession:	P27005,P31724,
UniProt Related Accession:	P27005
Molecular Weight:	10,295 Da
NCBI Full Name:	Protein S100-A8
NCBI Synonym Full Names:	S100 calcium binding protein A8 (calgranulin A)
NCBI Official Symbol:	S100a8
NCBI Official Synonym Symbols:	p8; B8Ag; CFAg; Caga; MRP8; CP-10; 60B8Ag; AI323541
NCBI Protein Information:	protein S100-A8
UniProt Protein Name:	Protein S100-A8
UniProt Synonym Protein Names:	Calgranulin-A; Chemotactic cytokine CP-10; Leukocyte L1 complex light chain; Migration inhibitory factor-related protein 8; MRP-8; p8; Pro-inflammatory S100 cytokine; S100 calcium-binding protein A8
Protein Family:	Protein
UniProt Gene Name:	S100a8

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

Step	Procedure
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	ELISA
S100A6 Antibody, Biotin conjugated	Mouse S100A8 (S100 Calcium Binding Protein A8) CLIA Kit
S100a8 Antibody	Mouse Protein S100-A8 (S100a8) ELISA Kit
S100a8 Antibody, HRP conjugated
S100a8 Antibody, FITC conjugated
S100A8 Antibody