人間 LAMC2 protein ELISA Kit (HUFI01352)- 高感度

商品名:	Human LAMC2 protein ELISA Kit
製品コード:	HUFI01352
サイズ:	96T
エイリアス:	LAMC2, Laminin subunit gamma-2, Laminin B2t chain, Ladsin 140 kDa subunit, Kalinin, nicein, epiligrin 100 kDa subunit, Kalinin subunit gamma, Nicein subunit gamma, Large adhesive scatter factor 140 kDa subunit, Laminin-5 subunit gamma, Cell-scattering factor 140 kDa subunit, CSF 140 kDa subunit, Epiligrin subunit gamma, LAMB2T, LAMNB2, B2T, BM600, CSF, EBR2, EBR2A
検出方法:	Sandwich ELISA, Double Antibody
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Human LAMC2 concentrations in serum plasma and other biological fluids.
感度:	0.094ng/ml
範囲:	0.156-10ng/ml
保管所:	4°C for 6 months
ノート:	For Research Use Only

回復:

Matrices listed below were spiked with certain level of Human LAMC2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human LAMC2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-101	95
EDTA plasma(n=5)	85-100	94
UFH plasma(n=5)	85-97	91

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human LAMC2 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)	87-99%	89-95%	85-103%
EDTA plasma(n=5)	82-96%	82-94%	85-97%
UFH plasma(n=5)	80-100%	86-95%	89-99%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

成分	量	保管所
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	-

必要なその他の材料と設備:

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

Q13753

UniProt Protein Function:	LAMC2: Binding to cells via a high affinity receptor, laminin is thought to mediate the attachment, migration and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components. Ladsin exerts cell- scattering activity toward a wide variety of cells, including epithelial, endothelial, and fibroblastic cells. Defects in LAMC2 are a cause of epidermolysis bullosa junctional Herlitz type (H-JEB); also known as junctional epidermolysis bullosa Herlitz-Pearson type. JEB defines a group of blistering skin diseases characterized by tissue separation which occurs within the dermo-epidermal basement membrane. H-JEB is a severe, infantile and lethal form. Death occurs usually within the first six months of life. Occasionally, children survive to teens. H-JEB is marked by bullous lesions at birth and extensive denudation of skin and mucous membranes that may be hemorrhagic. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted; Extracellular matrix; Motility/polarity/chemotaxis; Secreted, signal peptide Chromosomal Location of Human Ortholog: 1q25-q31 Cellular Component: extracellular space; laminin-5 complex; laminin-2 complex; membrane; perinuclear region of cytoplasm; extracellular region; cell cortex Molecular Function:heparin binding Biological Process: extracellular matrix disassembly; hemidesmosome assembly; epidermis development; extracellular matrix organization and biogenesis; cell adhesion Disease: Epidermolysis Bullosa, Junctional, Non-herlitz Type; Epidermolysis Bullosa, Junctional, Herlitz Type
NCBI Summary:	Laminins, a family of extracellular matrix glycoproteins, are the major noncollagenous constituent of basement membranes. They have been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis. Laminins, composed of 3 non identical chains: laminin alpha, beta and gamma (formerly A, B1, and B2, respectively), have a cruciform structure consisting of 3 short arms, each formed by a different chain, and a long arm composed of all 3 chains. Each laminin chain is a multidomain protein encoded by a distinct gene. Several isoforms of each chain have been described. Different alpha, beta and gamma chain isomers combine to give rise to different heterotrimeric laminin isoforms which are designated by Arabic numerals in the order of their discovery, i.e. alpha1beta1gamma1 heterotrimer is laminin 1. The biological functions of the different chains and trimer molecules are largely unknown, but some of the chains have been shown to differ with respect to their tissue distribution, presumably reflecting diverse functions in vivo. This gene encodes the gamma chain isoform laminin, gamma 2. The gamma 2 chain, formerly thought to be a truncated version of beta chain (B2t), is highly homologous to the gamma 1 chain; however, it lacks domain VI, and domains V, IV and III are shorter. It is expressed in several fetal tissues but differently from gamma 1, and is specifically localized to epithelial cells in skin, lung and kidney. The gamma 2 chain together with alpha 3 and beta 3 chains constitute laminin 5 (earlier known as kalinin), which is an integral part of the anchoring filaments that connect epithelial cells to the underlying basement membrane. The epithelium-specific expression of the gamma 2 chain implied its role as an epithelium attachment molecule, and mutations in this gene have been associated with junctional epidermolysis bullosa, a skin disease characterized by blisters due to disruption of the epidermal-dermal junction. Two transcript variants resulting from alternative splicing of the 3' terminal exon, and encoding different isoforms of gamma 2 chain, have been described. The two variants are differentially expressed in embryonic tissues, however, the biological significance of the two forms is not known. Transcript variants utilizing alternative polyA_signal have also been noted in literature. [provided by RefSeq, Aug 2011]
UniProt Code:	Q13753
NCBI GenInfo Identifier:	90185107
NCBI Gene ID:	3918
NCBI Accession:	Q13753.2
UniProt Secondary Accession:	Q13753,Q02536, Q02537, Q13752, Q14941, Q14DF7, Q2M1N2 Q5VYE8,
UniProt Related Accession:	Q13753
Molecular Weight:
NCBI Full Name:	Laminin subunit gamma-2
NCBI Synonym Full Names:	laminin, gamma 2
NCBI Official Symbol:	LAMC2
NCBI Official Synonym Symbols:	B2T; CSF; EBR2; BM600; EBR2A; LAMB2T; LAMNB2
NCBI Protein Information:	laminin subunit gamma-2; BM600-100kDa; laminin B2t chain; CSF 140 kDa subunit; nicein subunit gamma; kalinin subunit gamma; ladsin 140 kDa subunit; epiligrin subunit gamma; cell-scattering factor 140 kDa subunit; large adhesive scatter factor 140 kDa subunit
UniProt Protein Name:	Laminin subunit gamma-2
UniProt Synonym Protein Names:	Cell-scattering factor 140 kDa subunit; CSF 140 kDa subunit; Epiligrin subunit gamma; Kalinin subunit gamma; Kalinin/nicein/epiligrin 100 kDa subunit; Ladsin 140 kDa subunit; Laminin B2t chain; Laminin-5 subunit gamma; Large adhesive scatter factor 140 kDa subunit; Nicein subunit gamma
Protein Family:	Laminin
UniProt Gene Name:	LAMC2
UniProt Entry Name:	LAMC2_HUMAN

*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。

ウェルに加える前に、SABCワーキング溶液とTMB基質を37°Cで少なくとも30分間平衡化します。サンプルと試薬を希釈するときは、完全に均一に混合する必要があります。各テストの標準曲線をプロットすることをお勧めします。

ステップ	プロトコル
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.

ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。

サンプルタイプ	プロトコル
血清	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.
プラズマ	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.
尿および脳脊髄液	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.
細胞培養上清	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.
細胞溶解物	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.
組織ホモジネート	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.
組織溶解物	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.
母乳	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.