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C/EBP-beta (Phospho-Thr235/188) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01232
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Developmental Biology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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C/EBP-beta (Phospho-Thr235/188)Colorimetric Cell-Based ELISA Kit

The C/EBP Beta (phospho Thr235/188) Colorimetric Cell-Based ELISA Kit is a highly sensitive and specific assay designed for the precise detection of phosphorylated C/EBP Beta protein levels in cell lysates. This kit provides reliable and reproducible results, making it ideal for various research applications.C/EBP Beta is a transcription factor involved in regulating gene expression in response to various stimuli, including growth factors, cytokines, and stress signals. Phosphorylation of C/EBP Beta at Thr235/188 has been linked to its activation and role in cellular processes such as differentiation, proliferation, and inflammation.

This kit is essential for studying the role of phosphorylated C/EBP Beta in various cellular pathways and diseases, including cancer, metabolic disorders, and inflammatory conditions. With its high sensitivity and specificity, researchers can trust the accuracy of their results and further their understanding of the complex mechanisms involving C/EBP Beta.

Product Name:C/EBP-beta (Phospho-Thr235/188) Colorimetric Cell-Based ELISA
Product Code:CBCAB01232
ELISA Type:Cell-Based
Target:C/EBP-beta (Phospho-Thr235/188)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The C/EBP-beta (Phospho-Thr235/188) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect C/EBP-beta protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated C/EBP-beta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on C/EBP-beta phosphorylation.

Qualitative determination of C/EBP-beta (Phospho-Thr235/188) concentration is achieved by an indirect ELISA format. In essence, C/EBP-beta (Phospho-Thr235/188) is captured by C/EBP-beta (Phospho-Thr235/188)-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: 1051, UniProt ID: P17676, OMIM: 189965, Unigene: Hs.517106/Hs.701858/Hs.712709
Gene Symbol:CEBPB
Sub Type:Phospho
UniProt Protein Function:Important transcription factor regulating the expression of genes involved in immune and inflammatory responses (PubMed:1741402, PubMed:9374525, PubMed:12048245, PubMed:18647749). Plays also a significant role in adipogenesis, as well as in the gluconeogenic pathway, liver regeneration, and hematopoiesis. The consensus recognition site is 5'-T[TG]NNGNAA[TG]-3'. Its functional capacity is governed by protein interactions and post-translational protein modifications. During early embryogenesis, plays essential and redundant functions with CEBPA. Has a promitotic effect on many cell types such as hepatocytes and adipocytes but has an antiproliferative effect on T-cells by repressing MYC expression, facilitating differentiation along the T-helper 2 lineage. Binds to regulatory regions of several acute-phase and cytokines genes and plays a role in the regulation of acute-phase reaction and inflammation. Plays also a role in intracellular bacteria killing. During adipogenesis, is rapidly expressed and, after activation by phosphorylation, induces CEBPA and PPARG, which turn on the series of adipocyte genes that give rise to the adipocyte phenotype. The delayed transactivation of the CEBPA and PPARG genes by CEBPB appears necessary to allow mitotic clonal expansion and thereby progression of terminal differentiation (PubMed:20829347). Essential for female reproduction because of a critical role in ovarian follicle development. Restricts osteoclastogenesis.
NCBI Summary:This intronless gene encodes a transcription factor that contains a basic leucine zipper (bZIP) domain. The encoded protein functions as a homodimer but can also form heterodimers with CCAAT/enhancer-binding proteins alpha, delta, and gamma. Activity of this protein is important in the regulation of genes involved in immune and inflammatory responses, among other processes. The use of alternative in-frame AUG start codons results in multiple protein isoforms, each with distinct biological functions. [provided by RefSeq, Oct 2013]
UniProt Code:P17676
NCBI GenInfo Identifier:34223718
NCBI Gene ID:1051
NCBI Accession:P17676.2
UniProt Secondary Accession:P17676,Q96IH2, Q9H4Z5, A8K671,
UniProt Related Accession:P17676
Molecular Weight:15,938 Da
NCBI Full Name:CCAAT/enhancer-binding protein beta
NCBI Synonym Full Names:CCAAT/enhancer binding protein beta
NCBI Official Symbol:CEBPB  
NCBI Official Synonym Symbols:TCF5; IL6DBP; NF-IL6; C/EBP-beta  
NCBI Protein Information:CCAAT/enhancer-binding protein beta
UniProt Protein Name:CCAAT/enhancer-binding protein beta
UniProt Synonym Protein Names:Liver activator protein; LAP; Liver-enriched inhibitory protein; LIP; Nuclear factor NF-IL6
Protein Family:CCAAT/enhancer-binding protein
UniProt Gene Name:CEBPB  
UniProt Entry Name:CEBPB_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-C/EBP-beta (Phospho-Thr235/188) Antibody, Anti-C/EBP-beta 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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