IkappaB-alpha (Phospho-Tyr305) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00246
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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IkappaB-alpha (Phospho-Tyr305)Colorimetric Cell-Based ELISA Kit

The IKKalpha (Phospho-Tyr305) Colorimetric Cell-based ELISA Kit is a reliable and sensitive assay designed for the accurate detection of phosphorylated IKKalpha levels in cell lysates. This kit offers high specificity and reproducibility, making it ideal for a variety of research applications.IKKalpha is a key protein involved in the NF-kappaB signaling pathway, regulating inflammatory responses and cell survival. Phosphorylation of IKKalpha at Tyr305 plays a crucial role in modulating its activity and downstream signaling events.

Aberrant activation of this pathway has been implicated in various diseases, including cancer, inflammatory disorders, and autoimmune conditions.By accurately measuring phosphorylated IKKalpha levels, researchers can gain valuable insights into the mechanisms underlying these diseases and potentially identify new therapeutic targets. The IKKalpha (Phospho-Tyr305) Colorimetric Cell-based ELISA Kit provides a convenient and efficient tool for studying IKKalpha signaling pathways and evaluating potential therapeutic interventions.

Product Name:IkappaB-alpha (Phospho-Tyr305) Colorimetric Cell-Based ELISA
Product Code:CBCAB00246
ELISA Type:Cell-Based
Target:IkappaB-alpha (Phospho-Tyr305)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The IkappaB-alpha (Phospho-Tyr305) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect IkB-alpha protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IkB-alpha in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on IkB-alpha phosphorylation.

Qualitative determination of IkappaB-alpha (Phospho-Tyr305) concentration is achieved by an indirect ELISA format. In essence, IkappaB-alpha (Phospho-Tyr305) is captured by IkappaB-alpha (Phospho-Tyr305)-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: 4792, UniProt ID: P25963, OMIM: 164008/612132, Unigene: Hs.81328
Gene Symbol:NFKBIA
Sub Type:Phospho
UniProt Protein Function:Inhibits the activity of dimeric NF-kappa-B/REL complexes by trapping REL dimers in the cytoplasm through masking of their nuclear localization signals. On cellular stimulation by immune and proinflammatory responses, becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription.
NCBI Summary:This gene encodes a member of the NF-kappa-B inhibitor family, which contain multiple ankrin repeat domains. The encoded protein interacts with REL dimers to inhibit NF-kappa-B/REL complexes which are involved in inflammatory responses. The encoded protein moves between the cytoplasm and the nucleus via a nuclear localization signal and CRM1-mediated nuclear export. Mutations in this gene have been found in ectodermal dysplasia anhidrotic with T-cell immunodeficiency autosomal dominant disease. [provided by RefSeq, Aug 2011]
UniProt Code:P25963
NCBI GenInfo Identifier:126682
NCBI Gene ID:4792
NCBI Accession:P25963.1
UniProt Secondary Accession:P25963,B2R8L6,
UniProt Related Accession:P25963
Molecular Weight:35,609 Da
NCBI Full Name:NF-kappa-B inhibitor alpha
NCBI Synonym Full Names:NFKB inhibitor alpha
NCBI Official Symbol:NFKBIA  
NCBI Official Synonym Symbols:IKBA; MAD-3; NFKBI  
NCBI Protein Information:NF-kappa-B inhibitor alpha
UniProt Protein Name:NF-kappa-B inhibitor alpha
UniProt Synonym Protein Names:I-kappa-B-alpha; IkB-alpha; IkappaBalpha; Major histocompatibility complex enhancer-binding protein MAD3
Protein Family:NF-kappa-B inhibitor
UniProt Gene Name:NFKBIA  
UniProt Entry Name:IKBA_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-IkappaB-alpha (Phospho-Tyr305) Antibody, Anti-IkappaB-alpha 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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