Human Cytokeratin 6a / KRT6A ELISA Kit

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SKU:
HUFI00870
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P02538
Sensitivity:
18.75pg/ml
Range:
31.25-2000pg/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
KRT6A, KeRatin, type II cytoskeletal 6A, K6A, CK-6A, CK-6D, Type-II keRatin Kb6, KeRatin-6A, CytokeRatin-6A, CytokeRatin-6D, KRT6D
Reactivity:
Human
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Description

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Human Cytokeratin 6a / KRT6A ELISA Kit

Cytokeratin 6a is a member of the keratin family. The type II cytokeratins are basic or neutral proteins grouped in heterotypic keratin chains coexpressed during the differentiation of simple and stratified epithelial tissues. There are six distinct types of this type II cytokeratin (KRT6), with as many as six genes. The genes are activated in the filiform papillae of the tongue, the stratified epithelial lining of oral mucosa and esophagus, the outer root sheath of hair follicles, and glandular epithelia. The KRT6 gene in particular codes for the most prevalent form of the protein. Pachyonychia congenita has been linked to genetic changes in these genes. Peptides derived from the C-terminal portion of the protein have antibacterial activity. Pachyonychia congenita 3 and Pachyonychia Congenita 1 are examples of KRT6A-related illnesses.

Product Name:Human Cytokeratin 6a / KRT6A ELISA Kit
Product Code:HUFI00870
Size:96 Assays
Alias:KRT6A, KeRatin, type II cytoskeletal 6A, K6A, CK-6A, CK-6D, Type-II keRatin Kb6, KeRatin-6A, CytokeRatin-6A, CytokeRatin-6D, KRT6D
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human KRT6A concentrations in serum plasma and other biological fluids.
Sensitivity:18.75pg/ml
Range:31.25-2000pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human KRT6A and the recovery rates were calculated by comparing the measured value to the expected amount of Human KRT6A in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)91-9894
EDTA plasma(n=5)92-10399
UFH plasma(n=5)89-10297
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human KRT6A and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)88-105%94-102%88-99%
EDTA plasma(n=5)86-97%82-91%84-101%
UFH plasma(n=5)89-100%84-96%80-100%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotP02538
UniProt Protein Function:K6a: a type II cytoskeletal keratin. The keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into cytokeratins and hair keratins. There are two types of cytoskeletal and microfibrillar keratin: type I (acidic; 40-55 kDa) [K9 to K20] and type II (neutral to basic; 56-70 kDa) [K1 to K8]. Both a basic and an acidic keratin are required for filament assembly. Associates with K16 and/or -17.
UniProt Protein Details:Protein type:Cytoskeletal

Chromosomal Location of Human Ortholog: 12q13.13

Cellular Component: membrane; keratin filament; nucleus

Molecular Function:protein binding; structural constituent of cytoskeleton

Biological Process: wound healing; positive regulation of cell proliferation; morphogenesis of an epithelium; cell differentiation

Disease: Pachyonychia Congenita 3

NCBI Summary:The protein encoded by this gene is a member of the keratin gene family. The type II cytokeratins consist of basic or neutral proteins which are arranged in pairs of heterotypic keratin chains coexpressed during differentiation of simple and stratified epithelial tissues. As many as six of this type II cytokeratin (KRT6) have been identified; the multiplicity of the genes is attributed to successive gene duplication events. The genes are expressed with family members KRT16 and/or KRT17 in the filiform papillae of the tongue, the stratified epithelial lining of oral mucosa and esophagus, the outer root sheath of hair follicles, and the glandular epithelia. This KRT6 gene in particular encodes the most abundant isoform. Mutations in these genes have been associated with pachyonychia congenita. In addition, peptides from the C-terminal region of the protein have antimicrobial activity against bacterial pathogens. The type II cytokeratins are clustered in a region of chromosome 12q12-q13. [provided by RefSeq, Oct 2014]
UniProt Code:P02538
NCBI GenInfo Identifier:1346344
NCBI Gene ID:3853
NCBI Accession:P02538.3
UniProt Related Accession:P02538
Molecular Weight:60kDa
NCBI Full Name:Keratin, type II cytoskeletal 6A
NCBI Synonym Full Names:keratin 6A
NCBI Official Symbol:KRT6A  
NCBI Official Synonym Symbols:K6A; K6C; K6D; PC3; CK6A; CK6C; CK6D; CK-6C; CK-6E; KRT6C; KRT6D  
NCBI Protein Information:keratin, type II cytoskeletal 6A
UniProt Protein Name:Keratin, type II cytoskeletal 6A
UniProt Synonym Protein Names:Cytokeratin-6A; CK-6A; Cytokeratin-6D; CK-6D; Keratin-6A; K6A; Type-II keratin Kb6; Allergen: Hom s 5
UniProt Gene Name:KRT6A  
UniProt Entry Name:K2C6A_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.

StepProtocol
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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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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