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CDC16/APC6 (Phospho-Ser560) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01444
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Cycle
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

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CDC16/APC6 (Phospho-Ser560)Colorimetric Cell-Based ELISA Kit

The CDC16-APC6 Phospho-Ser560 Colorimetric Cell-Based ELISA Kit is specifically designed for the quantitative detection of phosphorylated CDC16-APC6 at serine 560 in cell lysates. This kit allows for the accurate measurement of phosphorylation levels, providing valuable insights into cell signaling pathways and regulatory mechanisms.CDC16-APC6 is a key component of the anaphase-promoting complex/cyclosome (APC/C) pathway, which regulates cell cycle progression and mitosis. Phosphorylation at serine 560 of CDC16-APC6 has been implicated in the control of cell division and chromosome segregation, making it an important target for research in cell biology and cancer biology.

With its high sensitivity and specificity, the CDC16-APC6 Phospho-Ser560 Colorimetric Cell-Based ELISA Kit offers reliable and reproducible results, making it an essential tool for studying the role of CDC16-APC6 phosphorylation in cellular processes and diseases. This kit is suitable for a wide range of research applications, providing valuable data for understanding cell cycle regulation and identifying potential therapeutic targets.

Product Name:CDC16/APC6 (Phospho-Ser560) Colorimetric Cell-Based ELISA
Product Code:CBCAB01444
ELISA Type:Cell-Based
Target:CDC16/APC6 (Phospho-Ser560)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The CDC16/APC6 (Phospho-Ser560) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CDC16/APC6 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CDC16/APC6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CDC16/APC6 phosphorylation.

Qualitative determination of CDC16/APC6 (Phospho-Ser560) concentration is achieved by an indirect ELISA format. In essence, CDC16/APC6 (Phospho-Ser560) is captured by CDC16/APC6 (Phospho-Ser560)-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: 8881, UniProt ID: Q13042, OMIM: 603461, Unigene: Hs.374127
Gene Symbol:CDC16
Sub Type:Phospho
UniProt Protein Function:CDC16: Component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle. The APC/C complex acts by mediating ubiquitination and subsequent degradation of target proteins: it mainly mediates the formation of 'Lys-11'-linked polyubiquitin chains and, to a lower extent, the formation of 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains. The APC/C is composed of at least 12 subunits. Interacts with PPP5C and CDC20. Belongs to the APC6/CDC16 family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell cycle regulation

Chromosomal Location of Human Ortholog: 13q34

Cellular Component: nucleoplasm; centrosome; anaphase-promoting complex; spindle microtubule; cytoplasm; spindle; cytosol

Molecular Function:protein binding

Biological Process: positive regulation of ubiquitin-protein ligase activity during mitotic cell cycle; cell proliferation; mitosis; negative regulation of ubiquitin-protein ligase activity during mitotic cell cycle; regulation of ubiquitin-protein ligase activity during mitotic cell cycle; cell division; anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process; mitotic cell cycle spindle assembly checkpoint; regulation of mitosis; mitotic cell cycle

NCBI Summary:This gene encodes a component protein of the APC complex, which is composed of eight proteins and functions as a protein ubiquitin ligase. The APC complex is a cyclin degradation system that governs exit from mitosis. Each component protein of the APC complex is highly conserved among eukaryotic organisms. This protein and two other APC complex proteins, CDC23 and CDC27, contain a tetratricopeptide repeat (TPR), a protein domain that may be involved in protein-protein interaction. Multiple alternatively spliced variants, encoding the same protein, have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:Q13042
NCBI GenInfo Identifier:37537763
NCBI Gene ID:8881
NCBI Accession:Q13042.2
UniProt Secondary Accession:Q13042,Q5T8C8, Q7Z651, Q96AE6, Q9Y564, A2A365,
UniProt Related Accession:Q13042
Molecular Weight:620
NCBI Full Name:Cell division cycle protein 16 homolog
NCBI Synonym Full Names:cell division cycle 16
NCBI Official Symbol:CDC16  
NCBI Official Synonym Symbols:APC6; CUT9; ANAPC6  
NCBI Protein Information:cell division cycle protein 16 homolog; CDC16Hs; cyclosome subunit 6; cell division cycle 16 homolog; anaphase-promoting complex subunit 6; anaphase-promoting complex, subunit 6
UniProt Protein Name:Cell division cycle protein 16 homolog
UniProt Synonym Protein Names:Anaphase-promoting complex subunit 6; APC6; CDC16 homolog; CDC16Hs; Cyclosome subunit 6
Protein Family:Cell division control protein
UniProt Gene Name:CDC16  
UniProt Entry Name:CDC16_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-CDC16/APC6 (Phospho-Ser560) Antibody, Anti-CDC16/APC6 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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