Product Name:	Human CAMK2B (Calcium/calmodulin-dependent protein kinase type II subunit beta) ELISA Kit
Product Code:	HUFI03476
Size:	96 Assays
Alias:	CaM kinase II subunit beta ELISA Kit, CAM2 ELISA Kit, CaMK II subunit beta ELISA Kit, CAMK2 ELISA Kit, CAMK2B ELISA Kit, CAMKB ELISA Kit, CaMKII beta ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human CAMK2B (Calcium/calmodulin-dependent protein kinase type II subunit beta) concentrations in serum plasma and other biological fluids.
Sensitivity:	< 0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human CAMK2B (Calcium/calmodulin-dependent protein kinase type II subunit beta) and the recovery rates were calculated by comparing the measured value to the expected amount of Human CAMK2B (Calcium/calmodulin-dependent protein kinase type II subunit beta) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-99	94
EDTA plasma(n=5)	88-100	96
heparin plasma(n=5)	86-101	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CAMK2B (Calcium/calmodulin-dependent protein kinase type II subunit beta) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-100%	86-101%	85-103%
EDTA plasma(n=5)	82-99%	83-101%	85-98%
UFH plasma(n=5)	81-99%	82-94%	80-98%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

UniProt Protein Function:	CAMK2B: a widely expressed protein kinase of the calcium/calmodulin-dependent protein kinase (CAMK) group. Functions autonomously after Ca2+/calmodulin-binding and autophosphorylation. Involved in dendritic spine and synapse formation, neuronal plasticity and regulation of sarcoplasmic reticulum Ca2+ transport in skeletal muscle. In neurons, plays an essential structural role in the reorganization of the actin cytoskeleton during plasticity by binding and bundling actin filaments in a kinase-independent manner. This structural function is required for correct targeting of CaMK2A, which acts downstream of NMDAR to promote dendritic spine and synapse formation and maintain synaptic plasticity which enables long-term potentiation (LTP) and hippocampus-dependent learning. In developing hippocampal neurons, promotes arborization of the dendritic tree and in mature neurons, promotes dendritic remodeling. Participates in the modulation of skeletal muscle function in response to exercise. In slow-twitch muscles, is involved in regulation of sarcoplasmic reticulum (SR) Ca2+ transport and in fast-twitch muscle participates in the control of Ca2+ release from the SR through phosphorylation of triadin, a ryanodine receptor-coupling factor, and phospholamban, an endogenous inhibitor of SERCA2. The holoenzyme is composed of 4 different chains: alpha (CAMK2A), beta (CAMK2B), gamma (CAMK2G), and delta (CAMK2D). The different isoforms assemble into homo- or heteromultimeric holoenzymes composed of 12 subunits with two hexameric rings stacked one on top of the other. Interacts with SYNGAP1 and CAMK2N2. Interacts with MPDZ. Expressed in adult and fetal brain. Expression is slightly lower in fetal brain. Expressed in skeletal muscle and induced during exercise. Eight isoforms of the human protein are produced by alternative splicing
UniProt Protein Details:	Protein type:EC 2.7.11.17; Kinase, protein; Protein kinase, CAMK; Protein kinase, Ser/Thr (non-receptor); CAMK group; CAMK2 family Chromosomal Location of Human Ortholog: 7p14.3-p14.1 Cellular Component: nucleoplasm; sarcoplasmic reticulum membrane; plasma membrane; microtubule organizing center; spindle midzone; cytosol Molecular Function:calmodulin binding; protein homodimerization activity; calmodulin-dependent protein kinase activity; actin binding; ATP binding Biological Process: regulation of long-term neuronal synaptic plasticity; regulation of skeletal muscle adaptation; protein amino acid autophosphorylation; cytokine and chemokine mediated signaling pathway; regulation of calcium ion transport; signal transduction; protein amino acid phosphorylation; regulation of synaptic transmission, cholinergic; inhibitory G-protein coupled receptor phosphorylation; synaptic transmission; peptidyl-serine phosphorylation; response to cadmium ion; calcium ion transport; regulation of synapse structural plasticity; neuromuscular process controlling balance; G1/S transition of mitotic cell cycle
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. Calcium signaling is crucial for several aspects of plasticity at glutamatergic synapses. In mammalian cells, the enzyme is composed of four different chains: alpha, beta, gamma, and delta. The product of this gene is a beta chain. It is possible that distinct isoforms of this chain have different cellular localizations and interact differently with calmodulin. Alternative splicing results in multiple transcript variants. [provided by RefSeq, May 2014]
UniProt Code:	Q13554
NCBI GenInfo Identifier:	334302890
NCBI Gene ID:	816
NCBI Accession:	Q13554.3
UniProt Secondary Accession:	Q13554,O95437, O95438, O95599, Q9UGH7, A4D2K0, A4D2K1 A4D2K2, A4D2K3, A4D2K4, A4D2K5, A4D2K6,
UniProt Related Accession:	Q13554
Molecular Weight:	664
NCBI Full Name:	Calcium/calmodulin-dependent protein kinase type II subunit beta
NCBI Synonym Full Names:	calcium/calmodulin-dependent protein kinase II beta
NCBI Official Symbol:	CAMK2B
NCBI Official Synonym Symbols:	CAM2; CAMK2; CAMKB
NCBI Protein Information:	calcium/calmodulin-dependent protein kinase type II subunit beta; caMK-II subunit beta; CaM-kinase II beta chain; CaM kinase II beta subunit; proline rich calmodulin-dependent protein kinase
UniProt Protein Name:	Calcium/calmodulin-dependent protein kinase type II subunit beta
Protein Family:	6-oxocamphor hydrolase
UniProt Gene Name:	CAMK2B
UniProt Entry Name:	KCC2B_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37 °C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37 °C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37 °C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.