Human MSH2 ELISA Kit (HUFI02038)- High Sensitivity

Product Name:	Human MSH2 ELISA Kit
Product Code:	HUFI02038
Size:	96 Assays
Alias:	MSH2, MSH2, COCA1, FCC1, HNPCC, HNPCC1, LCFS2, MutS protein homolog 2, LCFS2, DNA mismatch repair protein Msh2, hMSH2, HNPCC1mutS, E. coli homolog 2, colon cancer, nonpolyposis type 1, LCFS2, mutS homolog 2, colon cancer, nonpolyposis type 1, E. coli, MutS protein homolog 2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human MSH2 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 MSH2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human MSH2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-97	93
EDTA plasma(n=5)	86-105	98
UFH plasma(n=5)	96-104	99

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human MSH2 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)	85-105%	85-104%	85-99%
EDTA plasma(n=5)	83-101%	90-100%	86-96%
UFH plasma(n=5)	80-100%	84-95%	81-97%

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

P43246

UniProt Protein Function:	MSH2: Component of the post-replicative DNA mismatch repair system (MMR). Forms two different heterodimers: MutS alpha (MSH2- MSH6 heterodimer) and MutS beta (MSH2-MSH3 heterodimer) which binds to DNA mismatches thereby initiating DNA repair. When bound, heterodimers bend the DNA helix and shields approximately 20 base pairs. MutS alpha recognizes single base mismatches and dinucleotide insertion-deletion loops (IDL) in the DNA. MutS beta recognizes larger insertion-deletion loops up to 13 nucleotides long. After mismatch binding, MutS alpha or beta forms a ternary complex with the MutL alpha heterodimer, which is thought to be responsible for directing the downstream MMR events, including strand discrimination, excision, and resynthesis. ATP binding and hydrolysis play a pivotal role in mismatch repair functions. The ATPase activity associated with MutS alpha regulates binding similar to a molecular switch: mismatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts MutS alpha into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. This transition is crucial for mismatch repair. MutS alpha may also play a role in DNA homologous recombination repair. In melanocytes may modulate both UV-B-induced cell cycle regulation and apoptosis. Heterodimer consisting of MSH2-MSH6 (MutS alpha) or MSH2- MSH3 (MutS beta). Both heterodimer form a ternary complex with MutL alpha (MLH1-PMS1). Interacts with EXO1. Part of the BRCA1- associated genome surveillance complex (BASC), which contains BRCA1, MSH2, MSH6, MLH1, ATM, BLM, PMS2 and the RAD50-MRE11-NBS1 protein complex. This association could be a dynamic process changing throughout the cell cycle and within subnuclear domains. Interacts with ATR. Interacts with SLX4/BTBD12; this interaction is direct and links MutS beta to SLX4, a subunit of different structure-specific endonucleases. Interacts with SMARCAD1. Ubiquitously expressed. Belongs to the DNA mismatch repair MutS family.
UniProt Protein Details:	Protein type:Tumor suppressor; DNA-binding Chromosomal Location of Human Ortholog: 2p21 Cellular Component: membrane; MutSalpha complex; MutSbeta complex; nuclear chromosome, telomeric region; nucleoplasm Molecular Function:ADP binding; ATP binding; ATPase activity; centromeric DNA binding; dinucleotide insertion or deletion binding; dinucleotide repeat insertion binding; DNA binding; double-strand/single-strand DNA junction binding; double-stranded DNA binding; enzyme binding; four-way junction DNA binding; guanine/thymine mispair binding; loop DNA binding; magnesium ion binding; mismatched DNA binding; MutLalpha complex binding; oxidized purine DNA binding; protein binding; protein C-terminus binding; protein homodimerization activity; protein kinase binding; single guanine insertion binding; single thymine insertion binding; single-stranded DNA binding; Y-form DNA binding Biological Process: B cell differentiation; B cell mediated immunity; cell cycle arrest; determination of adult life span; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; DNA repair; double-strand break repair; germ cell development; in utero embryonic development; intra-S DNA damage checkpoint; isotype switching; maintenance of DNA repeat elements; male gonad development; meiotic gene conversion; mismatch repair; negative regulation of DNA recombination; negative regulation of meiotic recombination; negative regulation of neuron apoptosis; oxidative phosphorylation; positive regulation of helicase activity; postreplication repair; response to UV-B; response to X-ray; somatic hypermutation of immunoglobulin genes; somatic recombination of immunoglobulin gene segments Disease: Lynch Syndrome I; Mismatch Repair Cancer Syndrome; Muir-torre Syndrome
NCBI Summary:	This locus is frequently mutated in hereditary nonpolyposis colon cancer (HNPCC). When cloned, it was discovered to be a human homolog of the E. coli mismatch repair gene mutS, consistent with the characteristic alterations in microsatellite sequences (RER+ phenotype) found in HNPCC. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Apr 2012]
UniProt Code:	P43246
NCBI GenInfo Identifier:	1171032
NCBI Gene ID:	4436
NCBI Accession:	P43246.1
UniProt Secondary Accession:	P43246,O75488, B4E2Z2,
UniProt Related Accession:	P43246
Molecular Weight:	97,323 Da
NCBI Full Name:	DNA mismatch repair protein Msh2
NCBI Synonym Full Names:	mutS homolog 2
NCBI Official Symbol:	MSH2
NCBI Official Synonym Symbols:	FCC1; COCA1; HNPCC; LCFS2; HNPCC1
NCBI Protein Information:	DNA mismatch repair protein Msh2
UniProt Protein Name:	DNA mismatch repair protein Msh2
UniProt Synonym Protein Names:	MutS protein homolog 2
Protein Family:	DNA mismatch repair protein
UniProt Gene Name:	MSH2
UniProt Entry Name:	MSH2_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.

Human MSH2 ELISA Kit (HUFI02038)

Description