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ATF5 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00536
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

ATF5 Colorimetric Cell-Based ELISA Kit

The ATF5 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the precise quantification of Activating Transcription Factor 5 (ATF5) levels in cell lysates and culture media. This kit offers exceptional sensitivity and accuracy, ensuring dependable and consistent results for a variety of research purposes.ATF5 is a key transcription factor involved in regulating gene expression and cell differentiation.

Its dysregulation has been linked to various diseases, including cancer, neurodevelopmental disorders, and inflammatory conditions, making it a valuable target for therapeutic interventions. By providing a comprehensive understanding of ATF5 expression levels, this ELISA kit is an indispensable tool for advancing research in these fields and exploring potential treatment strategies.

Product Name:ATF5 Colorimetric Cell-Based ELISA
Product Code:CBCAB00536
ELISA Type:Cell-Based
Target:ATF5
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The ATF5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ATF5 protein expression profile in cells. The kit can be used for measuring the relative amounts of ATF5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ATF5.

Qualitative determination of ATF5 concentration is achieved by an indirect ELISA format. In essence, ATF5 is captured by ATF5-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: 22809, UniProt ID: Q9Y2D1, OMIM: 606398, Unigene: Hs.9754
Gene Symbol:ATF5
Sub Type:None
UniProt Protein Function:ATF-5: Transcriptional activator which binds the cAMP response element (CRE) (consensus: 5'-GTGACGT[AC][AG]-3'), a sequence present in many viral and cellular promoters and blocks the differentiation of neuroprogenitor cells into neurons. Its transcriptional activity is enhanced by CCND3 and slightly inhibited by CDK4. Belongs to the bZIP family.
UniProt Protein Details:

Protein type:DNA-binding

Chromosomal Location of Human Ortholog: 19q13.3

Cellular Component: centrosome; cytoplasm; nucleoplasm; nucleus

Molecular Function:chromatin binding; kinase binding; protein binding; sequence-specific DNA binding; transcription corepressor activity; transcription factor activity; tubulin binding

Biological Process: fat cell differentiation; granule cell precursor proliferation; negative regulation of apoptosis; negative regulation of cell proliferation; negative regulation of transcription, DNA-dependent; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of centrosome cycle; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent

UniProt Code:Q9Y2D1
NCBI GenInfo Identifier:308153647
NCBI Gene ID:22809
NCBI Accession:Q9Y2D1.4
UniProt Secondary Accession:Q9Y2D1,Q9BSA1, Q9UNQ3, B3KND3,
UniProt Related Accession:Q9Y2D1
Molecular Weight:30,674 Da
NCBI Full Name:Cyclic AMP-dependent transcription factor ATF-5
NCBI Synonym Full Names:activating transcription factor 5
NCBI Official Symbol:ATF5  
NCBI Official Synonym Symbols:ATFX; HMFN0395  
NCBI Protein Information:cyclic AMP-dependent transcription factor ATF-5
UniProt Protein Name:Cyclic AMP-dependent transcription factor ATF-5
UniProt Synonym Protein Names:Activating transcription factor 5; Transcription factor ATFx
Protein Family:Cyclic AMP-dependent transcription factor
UniProt Gene Name:ATF5  
UniProt Entry Name:ATF5_HUMAN
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-ATF5 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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