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IkappaB-epsilon (Phospho-Ser22) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01217
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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IkappaB-epsilon (Phospho-Ser22)Colorimetric Cell-Based ELISA Kit

The IKK Alpha (IKKα) Phospho-Ser176 Colorimetric Cell-Based ELISA Kit is specifically designed for the precise detection of IKK Alpha (IKKα) levels in cell lysates. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results, making it perfect for a variety of research applications.IKKα is a key protein involved in the regulation of inflammatory responses and immune cell activation. Its phosphorylation at serine 176 plays a crucial role in modulating its activity, making it a valuable biomarker for studying inflammatory diseases, including arthritis, autoimmune disorders, and cancer.

With the IKK Alpha (IKKα) Phospho-Ser176 Colorimetric Cell-Based ELISA Kit, researchers can easily measure IKKα phosphorylation levels in cell lysates, leading to a better understanding of its role in various physiological and pathological processes.

Product Name:IkappaB-epsilon (Phospho-Ser22) Colorimetric Cell-Based ELISA
Product Code:CBCAB01217
ELISA Type:Cell-Based
Target:IkappaB-epsilon (Phospho-Ser22)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The IkappaB-epsilon (Phospho-Ser22) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect IkB-epsilon protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated IkB-epsilon in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on IkB-epsilon phosphorylation.

Qualitative determination of IkappaB-epsilon (Phospho-Ser22) concentration is achieved by an indirect ELISA format. In essence, IkappaB-epsilon (Phospho-Ser22) is captured by IkappaB-epsilon (Phospho-Ser22)-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: 4794, UniProt ID: O00221, OMIM: 604548, Unigene: Hs.458276
Gene Symbol:NFKBIE
Sub Type:Phospho
UniProt Protein Function:IkB-epsilon: Inhibits NF-kappa-B by complexing with and trapping it in the cytoplasm. Inhibits DNA-binding of NF-kappa-B p50-p65 and p50-c-Rel complexes. Interacts with RELA, REL, NFKB1 nuclear factor NF-kappa-B p50 subunit and NFKB2 nuclear factor NF-kappa-B p52 subunit. Highly expressed in spleen, testis and lung, followed by kidney, pancreas, heart, placenta and brain. Also expressed in granulocytes and macrophages. Belongs to the NF-kappa-B inhibitor family.
UniProt Protein Details:

Protein type:DNA-binding; Inhibitor

Chromosomal Location of Human Ortholog: 6p21.1

Cellular Component: cytoplasm; cytosol

Molecular Function:protein binding

Biological Process: cytoplasmic sequestering of transcription factor

NCBI Summary:The protein encoded by this gene binds to components of NF-kappa-B, trapping the complex in the cytoplasm and preventing it from activating genes in the nucleus. Phosphorylation of the encoded protein targets it for destruction by the ubiquitin pathway, which activates NF-kappa-B by making it available to translocate to the nucleus. [provided by RefSeq, Sep 2011]
UniProt Code:O00221
NCBI GenInfo Identifier:206729919
NCBI Gene ID:4794
NCBI Accession:O00221.3
UniProt Secondary Accession:O00221,Q5T9V9,
UniProt Related Accession:O00221
Molecular Weight:52,864 Da
NCBI Full Name:NF-kappa-B inhibitor epsilon
NCBI Synonym Full Names:NFKB inhibitor epsilon
NCBI Official Symbol:NFKBIE  
NCBI Official Synonym Symbols:IKBE  
NCBI Protein Information:NF-kappa-B inhibitor epsilon
UniProt Protein Name:NF-kappa-B inhibitor epsilon
UniProt Synonym Protein Names:I-kappa-B-epsilon; IkB-E; IkB-epsilon; IkappaBepsilon
Protein Family:NF-kappa-B inhibitor
UniProt Gene Name:NFKBIE  
UniProt Entry Name:IKBE_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-epsilon (Phospho-Ser22) Antibody, Anti-IkappaB-epsilon 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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