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NEDD8 Colorimetric Cell-Based ELISA Kit (CBCAB00777)

SKU:
CBCAB00777
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Signal Transduction
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€769

Description

system_update_altDatasheetMSDS

NEDD8 Colorimetric Cell-Based ELISA Kit

The NEDD8 Colorimetric Cell-Based ELISA Kit from Assay Genie is a powerful tool for detecting NEDD8 levels in cell lysates. NEDD8 is a protein modifier that plays a critical role in regulating protein degradation and cell cycle progression. This kit offers high sensitivity and specificity, allowing for accurate and reliable measurement of NEDD8 levels in a variety of cell types. With its easy-to-use protocol and quick results, the NEDD8 Colorimetric Cell-Based ELISA Kit is ideal for researchers studying the role of NEDD8 in various cellular processes, including cell growth, differentiation, and response to stress.

By accurately measuring NEDD8 levels, researchers can gain valuable insights into the mechanisms underlying this important protein modifier and its impact on cellular function. Order your NEDD8 Colorimetric Cell-Based ELISA Kit today and take your research to the next level.

Product Name:NEDD8 Colorimetric Cell-Based ELISA
Product Code:CBCAB00777
ELISA Type:Cell-Based
Target:NEDD8
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The NEDD8 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect NEDD8 protein expression profile in cells. The kit can be used for measuring the relative amounts of NEDD8 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on NEDD8.

Qualitative determination of NEDD8 concentration is achieved by an indirect ELISA format. In essence, NEDD8 is captured by NEDD8-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: 4738, UniProt ID: Q15843, OMIM: 603171, Unigene: Hs.531064
Gene Symbol:NEDD8
Sub Type:None
UniProt Protein Function:NEDD8: a member of the ubiquitin family of proteins. Approximately 60% identical to ubiquitin protein. Activated by an E1-like complex, consisting of App-b1 and Uba3 and then linked to the E2-like enzyme, Ubc12. The major target protein modified by nedd8 is cullin-4a. Down-regulated during the development of brain.
UniProt Protein Details:

Protein type:Ubiquitin-like modifier

Chromosomal Location of Human Ortholog: 14q12

Cellular Component: cytosol; nucleus

Molecular Function:protein binding; ubiquitin protein ligase binding

Biological Process: regulation of transcription from RNA polymerase II promoter; ubiquitin-dependent protein catabolic process; protein localization; anatomical structure morphogenesis; protein neddylation; transforming growth factor beta receptor signaling pathway; protein modification process; proteolysis; response to organic cyclic substance

UniProt Code:Q15843
NCBI GenInfo Identifier:2833270
NCBI Gene ID:4738
NCBI Accession:Q15843.1
UniProt Secondary Accession:Q15843,Q3SXN8, Q6LES6,
UniProt Related Accession:Q15843
Molecular Weight:81
NCBI Full Name:NEDD8
NCBI Synonym Full Names:neural precursor cell expressed, developmentally down-regulated 8
NCBI Official Symbol:NEDD8  
NCBI Official Synonym Symbols:NEDD-8  
NCBI Protein Information:NEDD8; neddylin; ubiquitin-like protein Nedd8
UniProt Protein Name:NEDD8
UniProt Synonym Protein Names:Neddylin; Neural precursor cell expressed developmentally down-regulated protein 8; NEDD-8; Ubiquitin-like protein Nedd8
Protein Family:NEDD8
UniProt Gene Name:NEDD8  
UniProt Entry Name:NEDD8_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-NEDD8 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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