IKK-gamma (Phospho-Ser85) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB01470
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:IKK-gamma (Phospho-Ser85) Colorimetric Cell-Based ELISA
Product Code:CBCAB01470
ELISA Type:Cell-Based
Target:IKK-gamma (Phospho-Ser85)
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The IKK-gamma (Phospho-Ser85) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect IKK-gamma protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IKK-gamma in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on IKK-gamma phosphorylation.

Qualitative determination of IKK-gamma (Phospho-Ser85) concentration is achieved by an indirect ELISA format. In essence, IKK-gamma (Phospho-Ser85) is captured by IKK-gamma (Phospho-Ser85)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 8517, UniProt ID: Q9Y6K9, OMIM: 300248/300291/300301/300584/300636/300640/308300, Unigene: Hs.43505
Gene Symbol:IKBKG
Sub Type:Phospho
UniProt Protein Function:Regulatory subunit of the IKK core complex which phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. Its binding to scaffolding polyubiquitin seems to play a role in IKK activation by multiple signaling receptor pathways. However, the specific type of polyubiquitin recognized upon cell stimulation (either 'Lys-63'-linked or linear polyubiquitin) and its functional importance is reported conflictingly. Also considered to be a mediator for TAX activation of NF-kappa-B. Could be implicated in NF-kappa-B-mediated protection from cytokine toxicity. Essential for viral activation of IRF3. Involved in TLR3- and IFIH1-mediated antiviral innate response; this function requires 'Lys-27'-linked polyubiquitination.
NCBI Summary:This gene encodes the regulatory subunit of the inhibitor of kappaB kinase (IKK) complex, which activates NF-kappaB resulting in activation of genes involved in inflammation, immunity, cell survival, and other pathways. Mutations in this gene result in incontinentia pigmenti, hypohidrotic ectodermal dysplasia, and several other types of immunodeficiencies. A pseudogene highly similar to this locus is located in an adjacent region of the X chromosome. [provided by RefSeq, Mar 2016]
UniProt Code:Q9Y6K9
NCBI GenInfo Identifier:6685695
NCBI Gene ID:8517
NCBI Accession:Q9Y6K9.2
UniProt Secondary Accession:Q9Y6K9,Q7LBY6, Q7Z7F1,
UniProt Related Accession:Q9Y6K9
Molecular Weight:36,953 Da
NCBI Full Name:NF-kappa-B essential modulator
NCBI Synonym Full Names:inhibitor of nuclear factor kappa B kinase subunit gamma
NCBI Official Symbol:IKBKG  
NCBI Official Synonym Symbols:IP; IP1; IP2; FIP3; IKKG; IPD2; NEMO; FIP-3; Fip3p; IMD33; AMCBX1; IKKAP1; ZC2HC9; IKK-gamma  
NCBI Protein Information:NF-kappa-B essential modulator
UniProt Protein Name:NF-kappa-B essential modulator
UniProt Synonym Protein Names:FIP-3; IkB kinase-associated protein 1; IKKAP1; Inhibitor of nuclear factor kappa-B kinase subunit gamma; I-kappa-B kinase subunit gamma; IKK-gamma; IKKG; IkB kinase subunit gamma; NF-kappa-B essential modifier
UniProt Gene Name:IKBKG  
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-IKK-gamma (Phospho-Ser85) Antibody, Anti-IKK-gamma 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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