JMY Colorimetric Cell-Based ELISA

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SKU:
CBCAB00373
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
Frequently bought together:

Description

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JMY Colorimetric Cell-Based ELISA

The JMY Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the accurate measurement of protein levels in cell samples. This kit offers high sensitivity and specificity, providing researchers with reliable and reproducible results for a variety of research applications.With the ability to detect specific proteins within cell culture supernatants, this kit is ideal for studying the role of proteins in various cellular processes. Whether investigating cell signaling pathways, protein interactions, or cellular responses to stimuli, the JMY Colorimetric Cell-Based ELISA Kit provides valuable insights into cellular function.

By utilizing this advanced ELISA kit, researchers can gain a better understanding of the mechanisms underlying cell behavior and disease processes. With its ease of use and dependable performance, the JMY Colorimetric Cell-Based ELISA Kit is a valuable tool for advancing cell biology research.

Product Name:JMY Colorimetric Cell-Based ELISA
Product Code:CBCAB00373
ELISA Type:Cell-Based
Target:JMY
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of JMY concentration is achieved by an indirect ELISA format. In essence, JMY is captured by JMY-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: 133746, UniProt ID: Q8N9B5/A1L4P5, OMIM: 604279, Unigene: Hs.482605
Gene Symbol:JMY
Sub Type:None
UniProt Protein Function:JMY: Acts both as a nuclear p53/TP53-cofactor and a cytoplasmic regulator of actin dynamics depending on conditions. In nucleus, acts as a cofactor that increases p53/TP53 response via its interaction with p300/EP300. Increases p53/TP53-dependent transcription and apoptosis, suggesting an important role in p53/TP53 stress response such as DNA damage. In cytoplasm, acts as a nucleation-promoting factor for both branched and unbranched actin filaments. Activates the Arp2/3 complex to induce branched actin filament networks. Also catalyzes actin polymerization in the absence of Arp2/3, creating unbranched filaments. Contributes to cell motility by controlling actin dynamics. May promote the rapid formation of a branched actin network by first nucleating new mother filaments and then activating Arp2/3 to branch off these filaments. The p53/TP53-cofactor and actin activator activities are regulated via its subcellular location. Belongs to the JMY family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Apoptosis; Transcription, coactivator/corepressor

Chromosomal Location of Human Ortholog: 5q14.1

Cellular Component: leading edge; nucleoplasm; nucleus

Molecular Function:protein binding; transcription coactivator activity

Biological Process: cell cycle arrest; positive regulation of apoptosis; positive regulation of transcription factor activity; regulation of transcription from RNA polymerase II promoter

UniProt Code:Q8N9B5
NCBI GenInfo Identifier:172045777
NCBI Gene ID:133746
NCBI Accession:Q8N9B5.2
UniProt Secondary Accession:Q8N9B5,A1L4P5, B5MDS2, B5MDT0,
UniProt Related Accession:Q8N9B5
Molecular Weight:110,280 Da
NCBI Full Name:Junction-mediating and -regulatory protein
NCBI Synonym Full Names:junction mediating and regulatory protein, p53 cofactor
NCBI Official Symbol:JMY  
NCBI Official Synonym Symbols:WHAMM2; WHDC1L3  
NCBI Protein Information:junction-mediating and -regulatory protein
UniProt Protein Name:Junction-mediating and -regulatory protein
Protein Family:Junction-mediating and -regulatory protein
UniProt Gene Name:JMY  
UniProt Entry Name:JMY_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-JMY 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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