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PKD2 (Phospho-Ser876) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00243
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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PKD2 (Phospho-Ser876)Colorimetric Cell-Based ELISA Kit

The PKD2 Phospho-Ser876 Colorimetric Cell-Based ELISA Kit is a comprehensive assay designed to accurately measure levels of phosphorylated Ser876 site in the PKD2 protein in cell samples. This kit offers exceptional sensitivity and specificity, ensuring precise and consistent results for various research applications.PKD2 plays a critical role in cellular signaling pathways, influencing processes such as cell growth, proliferation, and differentiation. Phosphorylation at the Ser876 site is particularly important for regulating PKD2 activity and function. Dysfunction in PKD2 signaling has been linked to various diseases, including cancer, polycystic kidney disease, and cardiovascular disorders, making it a valuable marker for studying disease mechanisms and potential therapeutic interventions.

With the PKD2 Phospho-Ser876 Colorimetric Cell-Based ELISA Kit, researchers can accurately assess PKD2 phosphorylation levels in cell samples, providing valuable insights into biological processes and disease pathways. This kit is easy to use and delivers reliable results, making it an indispensable tool for investigating PKD2-related research questions.

Product Name:PKD2 (Phospho-Ser876) Colorimetric Cell-Based ELISA
Product Code:CBCAB00243
ELISA Type:Cell-Based
Target:PKD2 (Phospho-Ser876)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The PKD2 (Phospho-Ser876) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PKD2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PKD2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PKD2 phosphorylation.

Qualitative determination of PKD2 (Phospho-Ser876) concentration is achieved by an indirect ELISA format. In essence, PKD2 (Phospho-Ser876) is captured by PKD2 (Phospho-Ser876)-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: 25865, UniProt ID: Q9BZL6, OMIM: 607074, Unigene: Hs.466987
Gene Symbol:PRKD2
Sub Type:Phospho
UniProt Protein Function:PRKD2: a calcium-independent, phospholipid-dependent CAMK kinase of the PKD family. Activated by diacylglycerol and phorbol esters. may play a role in carcinoid tumor progression.
UniProt Protein Details:

Protein type:EC 2.7.11.13; Kinase, protein; Motility/polarity/chemotaxis; Protein kinase, Ser/Thr (non-receptor); Protein kinase, CAMK; CAMK group; PKD family

Chromosomal Location of Human Ortholog: 19q13.3

Cellular Component: cytoplasm; Golgi apparatus; nucleoplasm; nucleus; plasma membrane

Molecular Function:ATP binding; metal ion binding; protein binding; protein kinase activity; protein kinase C activity; protein serine/threonine kinase activity

Biological Process: activation of CREB transcription factor; activation of NF-kappaB transcription factor; adaptive immune response; angiogenesis; cell adhesion; cell death; peptidyl-serine phosphorylation; positive regulation of angiogenesis; positive regulation of blood vessel endothelial cell migration; positive regulation of cell adhesion; positive regulation of endothelial cell proliferation; positive regulation of fibroblast growth factor receptor signaling pathway; positive regulation of interleukin-2 production; positive regulation of interleukin-8 production; positive regulation of peptidyl-serine phosphorylation; positive regulation of T cell receptor signaling pathway; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of vascular endothelial growth factor receptor signaling pathway; protein amino acid autophosphorylation; protein amino acid phosphorylation; T cell receptor signaling pathway; vascular endothelial growth factor receptor signaling pathway

NCBI Summary:The protein encoded by this gene belongs to the protein kinase D (PKD) family of serine/threonine protein kinases. This kinase can be activated by phorbol esters as well as by gastrin via the cholecystokinin B receptor (CCKBR) in gastric cancer cells. It can bind to diacylglycerol (DAG) in the trans-Golgi network (TGN) and may regulate basolateral membrane protein exit from TGN. Alternative splicing results in multiple transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008]
UniProt Code:Q9BZL6
NCBI GenInfo Identifier:296434570
NCBI Gene ID:25865
NCBI Accession:Q9BZL6.2
UniProt Secondary Accession:Q9BZL6,Q8NCK8, Q8TB08, Q9P0T6, Q9Y3X8, M0R2R2,
UniProt Related Accession:Q9BZL6
Molecular Weight:
NCBI Full Name:Serine/threonine-protein kinase D2
NCBI Synonym Full Names:protein kinase D2
NCBI Official Symbol:PRKD2  
NCBI Official Synonym Symbols:PKD2; HSPC187; nPKC-D2  
NCBI Protein Information:serine/threonine-protein kinase D2
UniProt Protein Name:Serine/threonine-protein kinase D2
UniProt Synonym Protein Names:nPKC-D2
Protein Family:Serine/threonine-protein kinase
UniProt Gene Name:PRKD2  
UniProt Entry Name:KPCD2_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-PKD2 (Phospho-Ser876) Antibody, Anti-PKD2 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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