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Cyclin B1 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00609
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Cycle
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
Frequently bought together:

Description

system_update_altDatasheetMSDS

Cyclin B1 Colorimetric Cell-Based ELISA Kit

The Cyclin B1 Colorimetric Cell-Based ELISA Kit offered by Assay Genie is specifically designed for the accurate measurement of cyclin B1 levels in cell culture samples. This kit boasts high sensitivity and specificity, ensuring dependable and consistent results for a variety of research applications.Cyclin B1 is a key protein that regulates the cell cycle, specifically promoting the transition from the G2 phase to the M phase. Dysregulation of cyclin B1 has been implicated in various diseases, including cancer, making it a crucial biomarker for studying cell cycle progression and potential therapeutic targets.

With the Assay Genie Cyclin B1 Colorimetric Cell-Based ELISA Kit, researchers can confidently measure cyclin B1 levels in their cell culture samples, allowing for valuable insights into cell cycle dynamics and potential therapeutic interventions.

Product Name:Cyclin B1 Colorimetric Cell-Based ELISA
Product Code:CBCAB00609
ELISA Type:Cell-Based
Target:Cyclin B1
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Cyclin B1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Cyclin B1 protein expression profile in cells. The kit can be used for measuring the relative amounts of Cyclin B1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Cyclin B1.

Qualitative determination of Cyclin B1 concentration is achieved by an indirect ELISA format. In essence, Cyclin B1 is captured by Cyclin B1-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: 891, UniProt ID: P14635, OMIM: 123836, Unigene: Hs.23960
Gene Symbol:CCNB1
Sub Type:None
UniProt Protein Function:CCNB1: a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. Complexes with cdc2 to form the maturation-promoting factor (MPF). Two alternatively spliced isoforms have been found, a constitutively expressed form and a cell cycle-regulated form that is expressed predominantly during G2/M phase.
UniProt Protein Details:

Protein type:Cell cycle regulation; Activator

Chromosomal Location of Human Ortholog: 5q12

Cellular Component: nucleoplasm; spindle pole; centrosome; membrane; cytoplasm; nucleus; cytosol

Molecular Function:protein binding; patched binding; histone kinase activity; protein kinase binding

Biological Process: positive regulation of ubiquitin-protein ligase activity during mitotic cell cycle; positive regulation of histone phosphorylation; regulation of cell cycle; ventricular cardiac muscle cell development; positive regulation of attachment of spindle microtubules to kinetochore; mitotic nuclear envelope disassembly; positive regulation of mRNA 3'-end processing; positive regulation of mitotic cell cycle; positive regulation of fibroblast proliferation; negative regulation of protein amino acid phosphorylation; tissue regeneration; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; protein complex assembly; G2/M transition of mitotic cell cycle; positive regulation of cardiac muscle cell proliferation; response to drug; in utero embryonic development; oocyte maturation; mitotic metaphase plate congression; response to DDT; gut development; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; response to mechanical stimulus; cell division; spermatogenesis; regulation of cyclin-dependent protein kinase activity; mitotic cell cycle; mitotic spindle stabilization; G1/S transition of mitotic cell cycle

NCBI Summary:The protein encoded by this gene is a regulatory protein involved in mitosis. The gene product complexes with p34(cdc2) to form the maturation-promoting factor (MPF). Two alternative transcripts have been found, a constitutively expressed transcript and a cell cycle-regulated transcript, that is expressed predominantly during G2/M phase. The different transcripts result from the use of alternate transcription initiation sites. [provided by RefSeq, Jul 2008]
UniProt Code:P14635
NCBI GenInfo Identifier:116176
NCBI Gene ID:891
NCBI Accession:P14635.1
UniProt Secondary Accession:P14635,Q5TZP9, A8K066,
UniProt Related Accession:P14635
Molecular Weight:44,102 Da
NCBI Full Name:G2/mitotic-specific cyclin-B1
NCBI Synonym Full Names:cyclin B1
NCBI Official Symbol:CCNB1  
NCBI Official Synonym Symbols:CCNB  
NCBI Protein Information:G2/mitotic-specific cyclin-B1; G2/mitotic-specific cyclin B1
UniProt Protein Name:G2/mitotic-specific cyclin-B1
UniProt Gene Name:CCNB1  
UniProt Entry Name:CCNB1_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-Cyclin B1 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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