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CAMK2A/CAMK2D (Phospho-Thr286) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00037
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS
Product Name:CAMK2A/CAMK2D (Phospho-Thr286) Colorimetric Cell-Based ELISA
Product Code:CBCAB00037
ELISA Type:Cell-Based
Target:CAMK2A/CAMK2D (Phospho-Thr286)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The CAMK2A/CAMK2D (Phospho-Thr286) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CAMK2A/CAMK2D protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CAMK2A/CAMK2D in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CAMK2A/CAMK2D phosphorylation.

Qualitative determination of CAMK2A/CAMK2D (Phospho-Thr286) concentration is achieved by an indirect ELISA format. In essence, CAMK2A/CAMK2D (Phospho-Thr286) is captured by CAMK2A/CAMK2D (Phospho-Thr286)-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: 815/817, UniProt ID: Q9UQM7/Q13557, OMIM: 114078/607708, Unigene: Hs.143535/Hs.144114
Gene Symbol:CAMK2A/CAMK2D
Sub Type:Phospho
UniProt Protein Function:CAMK2A: a protein kinase of the CAMK2 family. A prominent kinase in the central nervous system that may function in long-term potentiation and neurotransmitter release. Member of the NMDAR signaling complex in excitatory synapses that may regulate NMDAR-dependent potentiation of the AMPAR and synaptic plasticity. The holoenzyme is composed of four different chains: alpha, beta, gamma, and delta. The different chains assemble into homo- or heteromultimeric holoenzymes composed of 8 to 12 subunits. May interact with BAALC, MPDZ, SYN1 and synGAP. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Protein kinase, CAMK; EC 2.7.11.17; Protein kinase, Ser/Thr (non-receptor); Kinase, protein; CAMK group; CAMK2 family

Chromosomal Location of Human Ortholog: 5q32

Cellular Component: cytosol; mitochondrion; nucleoplasm; nucleus; plasma membrane

Molecular Function:calmodulin binding; glutamate receptor binding; kinase activity; protein binding; protein homodimerization activity; protein serine/threonine kinase activity; Ras guanyl-nucleotide exchange factor activity

Biological Process: activation of NF-kappaB transcription factor; calcium ion transport; G1/S transition of mitotic cell cycle; MAPKKK cascade; peptidyl-serine phosphorylation; positive regulation of calcium ion transport; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of neuronal synaptic plasticity; regulation of neurotransmitter secretion; Wnt receptor signaling pathway, calcium modulating pathway

NCBI Summary:The product of this gene belongs to the serine/threonine protein kinases family, and to the Ca(2+)/calmodulin-dependent protein kinases subfamily. Calcium signaling is crucial for several aspects of plasticity at glutamatergic synapses. This calcium calmodulin-dependent protein kinase is composed of four different chains: alpha, beta, gamma, and delta. The alpha chain encoded by this gene is required for hippocampal long-term potentiation (LTP) and spatial learning. In addition to its calcium-calmodulin (CaM)-dependent activity, this protein can undergo autophosphorylation, resulting in CaM-independent activity. Two transcript variants encoding distinct isoforms have been identified for this gene. [provided by RefSeq, Nov 2008]
UniProt Code:Q9UQM7
NCBI GenInfo Identifier:296434552
NCBI Gene ID:815
NCBI Accession:Q9UQM7.2
UniProt Secondary Accession:Q9UQM7,Q9UL21, Q9Y2H4, Q9Y352,
UniProt Related Accession:Q9UQM7
Molecular Weight:55,320 Da
NCBI Full Name:Calcium/calmodulin-dependent protein kinase type II subunit alpha
NCBI Synonym Full Names:calcium/calmodulin dependent protein kinase II alpha
NCBI Official Symbol:CAMK2A  
NCBI Official Synonym Symbols:CAMKA  
NCBI Protein Information:calcium/calmodulin-dependent protein kinase type II subunit alpha
UniProt Protein Name:Calcium/calmodulin-dependent protein kinase type II subunit alpha
Protein Family:Calcium/calmodulin-dependent protein kinase
UniProt Gene Name:CAMK2A  
UniProt Entry Name:KCC2A_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-CAMK2A/CAMK2D (Phospho-Thr286) Antibody, Anti-CAMK2A/CAMK2D 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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