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CaMK4 (Phospho-Thr196/200) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01395
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

CaMK4 (Phospho-Thr196/200)Colorimetric Cell-Based ELISA Kit

The CAMK4 Phospho-Thr196-200 Colorimetric Cell-Based ELISA Kit is specially designed for the accurate detection and quantification of phosphorylated CAMK4 at threonine residues 196 and 200 in cell lysates. This kit offers exceptional sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.CAMK4, also known as calcium/calmodulin-dependent protein kinase IV, plays a crucial role in various cellular processes, including gene expression, neuronal activity, and synaptic plasticity. Phosphorylation of CAMK4 at threonine residues 196 and 200 can modulate its activity and function, making it an important target for studying signal transduction pathways and cellular responses.

This colorimetric cell-based ELISA kit provides a convenient and efficient method for analyzing CAMK4 phosphorylation in cell samples, offering insights into intracellular signaling pathways and potential therapeutic targets. Researchers can utilize this kit to investigate the role of CAMK4 in various physiological and pathological conditions, such as neuronal development, learning and memory, and neurodegenerative disorders.

Product Name:CaMK4 (Phospho-Thr196/200) Colorimetric Cell-Based ELISA
Product Code:CBCAB01395
ELISA Type:Cell-Based
Target:CaMK4 (Phospho-Thr196/200)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The CaMK4 (Phospho-Thr196/200) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CaMK4 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CaMK4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CaMK4 phosphorylation.

Qualitative determination of CaMK4 (Phospho-Thr196/200) concentration is achieved by an indirect ELISA format. In essence, CaMK4 (Phospho-Thr196/200) is captured by CaMK4 (Phospho-Thr196/200)-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: 814, UniProt ID: Q16566, OMIM: 114080, Unigene: Hs.591269
Gene Symbol:CAMK4
Sub Type:Phospho
UniProt Protein Function:CAMK4: a protein kinase of the CAMK1 family. Implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. Two alternatively sliced isoforms have been described.
UniProt Protein Details:

Protein type:Protein kinase, Ser/Thr (non-receptor); Protein kinase, CAMK; EC 2.7.11.17; Kinase, protein; CAMK group; CAMK1 family

Chromosomal Location of Human Ortholog: 5q21.3

Cellular Component: cytoplasm; cytosol; nucleolus; nucleoplasm

Molecular Function:ATP binding; calcium-dependent protein serine/threonine kinase activity; calmodulin binding; calmodulin-dependent protein kinase activity

Biological Process: adaptive immune response; epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; inflammatory response; innate immune response; long-term memory; myeloid dendritic cell cytokine production; myeloid dendritic cell differentiation; nerve growth factor receptor signaling pathway; nerve-nerve synaptic transmission; nucleocytoplasmic transport; peptidyl-serine phosphorylation; phospholipase C activation; positive regulation of protein export from nucleus; positive regulation of transcription, DNA-dependent; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of osteoclast differentiation; regulation of T cell differentiation in the thymus; signal transduction; synaptic transmission

NCBI Summary:The product of this gene belongs to the serine/threonine protein kinase family, and to the Ca(2+)/calmodulin-dependent protein kinase subfamily. This enzyme is a multifunctional serine/threonine protein kinase with limited tissue distribution, that has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. [provided by RefSeq, Jul 2008]
UniProt Code:Q16566
NCBI GenInfo Identifier:2499586
NCBI Gene ID:814
NCBI Accession:Q16566.1
UniProt Secondary Accession:Q16566,D3DSZ7,
UniProt Related Accession:Q16566
Molecular Weight:51,926 Da
NCBI Full Name:Calcium/calmodulin-dependent protein kinase type IV
NCBI Synonym Full Names:calcium/calmodulin-dependent protein kinase IV
NCBI Official Symbol:CAMK4  
NCBI Official Synonym Symbols:caMK; CaMK IV; CaMK-GR  
NCBI Protein Information:calcium/calmodulin-dependent protein kinase type IV
UniProt Protein Name:Calcium/calmodulin-dependent protein kinase type IV
UniProt Synonym Protein Names:CaM kinase-GR
Protein Family:Calcium/calmodulin-dependent protein kinase
UniProt Gene Name:CAMK4  
UniProt Entry Name:KCC4_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-CaMK4 (Phospho-Thr196/200) Antibody, Anti-CaMK4 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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