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Annexin A6 Colorimetric Cell-Based ELISA Kit (CBCAB00526)

SKU:
CBCAB00526
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Detection Method:
Colorimetric
€769

Description

system_update_altDatasheet

Annexin A6 Colorimetric Cell-Based ELISA Kit

The Annexin A6 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers studying Annexin A6 levels in cellular samples. This kit offers high sensitivity and specificity, ensuring accurate and reliable results for a variety of research applications.Annexin A6 is a protein involved in cellular processes such as membrane trafficking and cell signaling. Abnormal levels of Annexin A6 have been linked to various diseases including cancer, cardiovascular diseases, and neurodegenerative disorders, making it an important biomarker for studying these conditions and developing potential treatments.

With the Annexin A6 Colorimetric Cell-Based ELISA Kit, researchers can confidently measure Annexin A6 levels in cell lysates, culture supernatants, and other samples with precision and convenience. This kit is a valuable resource for those investigating the role of Annexin A6 in cellular function and disease pathogenesis.

Product Name:Annexin A6 Colorimetric Cell-Based ELISA
Product Code:CBCAB00526
ELISA Type:Cell-Based
Target:Annexin A6
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Annexin A6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Annexin A6 protein expression profile in cells. The kit can be used for measuring the relative amounts of Annexin A6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Annexin A6.

Qualitative determination of Annexin A6 concentration is achieved by an indirect ELISA format. In essence, Annexin A6 is captured by Annexin A6-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 309, UniProt ID: P08133, OMIM: 114070, Unigene: Hs.412117
Gene Symbol:ANXA6
Sub Type:None
NCBI Summary:Annexin VI belongs to a family of calcium-dependent membrane and phospholipid binding proteins. Several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. The annexin VI gene is approximately 60 kbp long and contains 26 exons. It encodes a protein of about 68 kDa that consists of eight 68-amino acid repeats separated by linking sequences of variable lengths. It is highly similar to human annexins I and II sequences, each of which contain four such repeats. Annexin VI has been implicated in mediating the endosome aggregation and vesicle fusion in secreting epithelia during exocytosis. Alternatively spliced transcript variants have been described. [provided by RefSeq, Aug 2010]
UniProt Code:P08133
NCBI GenInfo Identifier:71773329
NCBI Gene ID:309
NCBI Accession:NP_001146.2
UniProt Secondary Accession:P08133,Q6ZT79, B7Z8A7, D3DQH4, E9PGK1,
UniProt Related Accession:P08133
Molecular Weight:75,873 Da
NCBI Full Name:annexin A6 isoform 1
NCBI Synonym Full Names:annexin A6
NCBI Official Symbol:ANXA6  
NCBI Official Synonym Symbols:ANX6; CBP68  
NCBI Protein Information:annexin A6; p68; p70; CPB-II; annexin-6; calelectrin; lipocortin VI; calphobindin II; calphobindin-II; chromobindin-20; annexin VI (p68); 67 kDa calelectrin; calcium-binding protein p68
UniProt Protein Name:
UniProt Gene Name:
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)
  • Deionized or sterile water

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

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.Add 50 µL of 1x primary antibodies (Anti-Annexin A6 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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