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Human ATF6 / Activating Transcription Factor 6 ELISA Kit

SKU:
HUFI02241
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P18850
Sensitivity:
46.875pg/ml
Range:
78.125-5000pg/ml
ELISA Type:
Sandwich
Synonyms:
ATF-6
Reactivity:
Human
Research Area:
Epigenetics and Nuclear Signaling
€599
Frequently bought together:

Description

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Human ATF6 / Activating Transcription Factor 6 ELISA

ATF6 (Activating Transcription Factor 6) protein is a member of the basic leucine zipper family of transcription factors, and is involved in the unfolded protein response. The ATF6 protein is expressed in the endoplasmic reticulum and is activated by the accumulation of unfolded proteins. Under normal conditions, the ATF6 protein is inactive, but it is activated when unfolded proteins accumulate in the ER. The ATF6 protein plays an important role in protecting cells from damage caused by unfolded proteins.

Product Name:Human ATF6 / Activating Transcription Factor 6 ELISA Kit
Product Code:HUFI02241
Size:96 Assays
Alias:ATF-6
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human ATF-6 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 ATF-6 and the recovery rates were calculated by comparing the measured value to the expected amount of Human ATF-6 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)86-10395
EDTA plasma(n=5)85-10595
UFH plasma(n=5)94-10399
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ATF-6 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-100%87-102%91-101%
EDTA plasma(n=5)87-98%82-99%89-99%
UFH plasma(n=5)80-100%82-99%81-100%
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
UniprotP18850
UniProt Protein Function:ATF6A: Transcription factor that acts during endoplasmic reticulum stress by activating unfolded protein response target genes. Binds DNA on the 5'-CCAC[GA]-3'half of the ER stress response element (ERSE) (5'-CCAAT-N(9)-CCAC[GA]-3') and of ERSE II (5'-ATTGG-N-CCACG-3'). Binding to ERSE requires binding of NF-Y to ERSE. Could also be involved in activation of transcription by the serum response factor. Belongs to the bZIP family. ATF subfamily.
UniProt Protein Details:Protein type:Transcription factor; Membrane protein, integral

Chromosomal Location of Human Ortholog: 1q23.3

Cellular Component: endoplasmic reticulum; endoplasmic reticulum membrane; Golgi apparatus; Golgi membrane; integral to endoplasmic reticulum membrane; membrane; nuclear envelope; nucleoplasm; nucleus

Molecular Function:identical protein binding; protein binding; protein heterodimerization activity; transcription coactivator activity; transcription factor activity

Biological Process: eye development; positive regulation of transcription of target genes involved in unfolded protein response; protein folding; regulation of transcription from RNA polymerase II promoter; response to stress; signal transduction; unfolded protein response; visual perception

Disease: Achromatopsia 7

NCBI Summary:This gene encodes a transcription factor that activates target genes for the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. Although it is a transcription factor, this protein is unusual in that it is synthesized as a transmembrane protein that is embedded in the ER. It functions as an ER stress sensor/transducer, and following ER stress-induced proteolysis, it functions as a nuclear transcription factor via a cis-acting ER stress response element (ERSE) that is present in the promoters of genes encoding ER chaperones. This protein has been identified as a survival factor for quiescent but not proliferative squamous carcinoma cells. There have been conflicting reports about the association of polymorphisms in this gene with diabetes in different populations, but another polymorphism has been associated with increased plasma cholesterol levels. This gene is also thought to be a potential therapeutic target for cystic fibrosis. [provided by RefSeq, Aug 2011]
UniProt Code:P18850
NCBI GenInfo Identifier:66774203
NCBI Gene ID:22926
NCBI Accession:P18850.3
UniProt Secondary Accession:P18850,O15139, Q5VW62, Q6IPB5, Q9UEC9,
UniProt Related Accession:P18850
Molecular Weight:74,585 Da
NCBI Full Name:Cyclic AMP-dependent transcription factor ATF-6 alpha
NCBI Synonym Full Names:activating transcription factor 6
NCBI Official Symbol:ATF6
NCBI Official Synonym Symbols:ACHM7; ATF6A
NCBI Protein Information:cyclic AMP-dependent transcription factor ATF-6 alpha
UniProt Protein Name:Cyclic AMP-dependent transcription factor ATF-6 alpha
UniProt Synonym Protein Names:Activating transcription factor 6 alpha; ATF6-alpha
Protein Family:Cyclic AMP-dependent transcription factor
UniProt Gene Name:ATF6
UniProt Entry Name:ATF6A_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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