null

NAB2 Colorimetric Cell-Based ELISA

SKU:
CBCAB01116
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

NAB2 Colorimetric Cell-Based ELISA

The NAB2 Colorimetric Cell-Based ELISA Kit is a highly sensitive and reliable tool for the detection of NAB2 levels in cell culture supernatants. This kit offers exceptional specificity and precision, allowing for accurate and reproducible results across various research applications.NAB2, also known as NGFI-A Binding Protein 2, is a key transcriptional regulator involved in various cellular processes such as cell growth, differentiation, and apoptosis. Dysregulation of NAB2 has been implicated in numerous diseases, including cancer and neurological disorders, making it a critical target for therapeutic interventions.

With the NAB2 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of NAB2 in disease pathogenesis and potentially uncover new treatment strategies. This kit is a valuable tool for advancing scientific knowledge and driving innovation in the field of cell biology and molecular research.

Product Name:NAB2 Colorimetric Cell-Based ELISA
Product Code:CBCAB01116
ELISA Type:Cell-Based
Target:NAB2
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The NAB2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NAB2 protein expression profile in cells. The kit can be used for measuring the relative amounts of NAB2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NAB2.

Qualitative determination of NAB2 concentration is achieved by an indirect ELISA format. In essence, NAB2 is captured by NAB2-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: 4665, UniProt ID: Q15742, OMIM: 602381, Unigene: Hs.159223
Gene Symbol:NAB2
Sub Type:None
UniProt Protein Function:NAB2: Acts as a transcriptional repressor for zinc finger transcription factors EGR1 and EGR2. Isoform 2 lacks repression ability. Belongs to the NAB family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Chromosomal Location of Human Ortholog: 12q13.3

Cellular Component: nucleus

Molecular Function:protein binding; transcription corepressor activity

Biological Process: cell proliferation; negative regulation of transcription from RNA polymerase III promoter; nervous system development

NCBI Summary:This gene encodes a member of the family of NGFI-A binding (NAB) proteins, which function in the nucleus to repress transcription induced by some members of the EGR (early growth response) family of transactivators. NAB proteins can homo- or hetero-multimerize with other EGR or NAB proteins through a conserved N-terminal domain, and repress transcription through two partially redundant C-terminal domains. Transcriptional repression by the encoded protein is mediated in part by interactions with the nucleosome remodeling and deactylase (NuRD) complex. Alternatively spliced transcript variants have been described, but their biological validity has not been determined. [provided by RefSeq, Jul 2008]
UniProt Code:Q15742
NCBI GenInfo Identifier:12643729
NCBI Gene ID:4665
NCBI Accession:Q15742.1
UniProt Secondary Accession:Q15742,O76006, Q14797, B2RAK3,
UniProt Related Accession:Q15742
Molecular Weight:49,748 Da
NCBI Full Name:NGFI-A-binding protein 2
NCBI Synonym Full Names:NGFI-A binding protein 2
NCBI Official Symbol:NAB2  
NCBI Official Synonym Symbols:MADER  
NCBI Protein Information:NGFI-A-binding protein 2
UniProt Protein Name:NGFI-A-binding protein 2
UniProt Synonym Protein Names:EGR-1-binding protein 2; Melanoma-associated delayed early response protein; Protein MADER
Protein Family:NGFI-A-binding protein
UniProt Gene Name:NAB2  
UniProt Entry Name:NAB2_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-NAB2 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.)

Related Products