Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit

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SKU:
RTFI00410
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit - Information

The ELISA Genie Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit can assay for Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit - Data

Description

p35 is a neuron specific activator of CDK5. The complex p35/CDK5 is required for neurite outgrowth and cortical lamination. Involved in dendritic spine morphogenesis by mediating the EFNA1-EPHA4 signaling. Activator of TPKII. The complex p35/CDK5 participates in the regulation of the circadian clock by modulating the function of CLOCK protein: phosphorylates CLOCK at 'Thr-451' and 'Thr-461' and regulates the transcriptional activity of the CLOCK-ARNTL/BMAL1 heterodimer in association with altered stability and subcellular distribution.

Post-Translational Modification

The p35 form is proteolytically cleaved by calpain, giving rise to the p25 form. P35 has a 5 to 10 fold shorter half-life compared to p25. The conversion results in deregulation of the CDK5 kinase: p25/CDK5 kinase displays an increased and altered tau phosphorylation in comparison to the p35/CDK5 kinase in vivo. Myristoylated. A proper myristoylation signal is essential for the proper distribution of p35. Ubiquitinated. Degradation of p35 by proteasome results in down-regulation of CDK5 activity. During this process, CDK5 phosphorylates p35 and induces its ubiquitination and subsequent degradation. Phosphorylation at Ser-8 and Thr-138 by CDK5 prevents calpain-mediated proteolysis.

Uniprot ID P61810
Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Cdk5r1 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.156-10ng/ml

Sensitivity

< 0.094ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Cdk5r1 and the recovery rates were calculated by comparing the measured value to the expected amount of Cdk5r1 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 96-105 99
EDTA plasma(n=5) 91-105 96
UFH plasma(n=5) 85-105 94
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Cdk5r1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 89-105% 86-102% 85-104% 89-100%
EDTA plasma(n=5) 83-99% 82-93% 84-100% 85-97%
UFH plasma(n=5) 85-97% 83-99% 80-95% 80-89%
CV(%)

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

Note

For Research Use Only

Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit Protocol

The below protocol is a sample protocol for Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 present in their sample.

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.

Sandwich Protocol

Sandwich ELISA Protocol

Kit 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.

Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Rat CDK5R1 / Cyclin-dependent kinase 5 activator 1 ELISA Kit Protein Information

UniProt Protein Function:CDK5R1: activator of cyclin-dependent kinase 5 (CDK5). Required for proper development of the central nervous system. The p35 form of this protein is cleaved by calpain, generating a p25 form, resulting in the prolonged activation and relocalization of the CDK5 from the cell periphery to nuclear and perinuclear regions. The p25 form accumulates in the brain neurons of patients with Alzheimer's disease, leading to aberrantly phosphorylated forms of the microtubule-associated protein tau, which contributes to Alzheimer's disease.
UniProt Protein Details:

Protein type:Protein kinase, regulatory subunit

Chromosomal Location of Human Ortholog: 10q26

Cellular Component: axon; cell soma; contractile fiber; cyclin-dependent protein kinase 5 activator complex; cytoplasm; dendrite; dendritic spine; growth cone; intracellular membrane-bound organelle; membrane; neuromuscular junction; nucleoplasm; nucleus; perinuclear region of cytoplasm; postsynaptic density

Molecular Function:cadherin binding; calcium ion binding; cyclin-dependent protein kinase 5 activator activity; ephrin receptor binding; kinase activity; protein binding; protein kinase binding; protein serine/threonine kinase activator activity

Biological Process: acetylcholine receptor signaling, muscarinic pathway; axon extension; axon guidance; axonal fasciculation; brain development; cerebellum development; cerebral cortex radially oriented cell migration; ephrin receptor signaling pathway; hippocampus development; ionotropic glutamate receptor signaling pathway; layer formation in the cerebral cortex; negative regulation of axon extension; negative regulation of transcription, DNA-dependent; neurite development; neuron adhesion; neuron differentiation; neuron migration; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; positive regulation of neuron apoptosis; positive regulation of protein kinase activity; regulation of actin cytoskeleton organization and biogenesis; serine phosphorylation of STAT protein; superior olivary nucleus maturation

NCBI Summary:kinase that phosphorylates neurofilament protein (NF-H)and intermediate filament protein nestin [RGD, Feb 2006]
UniProt Code:P61810
NCBI GenInfo Identifier:16758762
NCBI Gene ID:116671
NCBI Accession:NP_446343.1
UniProt Secondary Accession:P61810,Q62938,
UniProt Related Accession:P61810
Molecular Weight:34,031 Da
NCBI Full Name:cyclin-dependent kinase 5 activator 1
NCBI Synonym Full Names:cyclin-dependent kinase 5 regulatory subunit 1
NCBI Official Symbol:Cdk5r1  
NCBI Official Synonym Symbols:p35; Cdk5r  
NCBI Protein Information:cyclin-dependent kinase 5 activator 1
UniProt Protein Name:Cyclin-dependent kinase 5 activator 1
UniProt Synonym Protein Names:Cyclin-dependent kinase 5 regulatory subunit 1; TPKII regulatory subunit
Protein Family:Cyclin-dependent kinase 5 activator
UniProt Gene Name:Cdk5r1  
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Additional Information

Reactivity:
Rat
ELISA Type:
Sandwich
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