The Mouse Cyclin-Dependent Kinase Inhibitor 2A Isoforms 1/2 (CDKN2A) ELISA Kit is specifically designed for the precise quantification of CDKN2A levels in mouse serum, plasma, and tissue lysates. This kit offers exceptional sensitivity and accuracy, ensuring robust and consistent results that are suitable for various research applications.CDKN2A, also known as p16 or p14ARF, is a key regulator of cell cycle progression by inhibiting cyclin-dependent kinases. Dysregulation of CDKN2A has been implicated in various diseases, including cancer and aging-related disorders, making it a valuable marker for studying these conditions and potential therapeutic interventions.
With the Mouse Cyclin-Dependent Kinase Inhibitor 2A Isoforms 1/2 ELISA Kit, researchers can effectively measure CDKN2A levels in mouse models, advancing our understanding of cell cycle control and disease mechanisms. This kit will undoubtedly facilitate groundbreaking discoveries in the field of molecular biology and drug development.
Cyclin-dependent kinase 4 inhibitor A, p16-INK4a, CDK4I, p16-INK4, P16ink4a
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
1.56-100ng/mL
Sensitivity:
0.88ng/mL
Intra CV:
Provided with the Kit
Inter CV:
Provided with the Kit
Linearity:
Provided with the Kit
Recovery:
Provided with the Kit
Function:
Acts as a negative regulator of the proliferation of normal cells by interacting strongly with CDK4 and CDK6. This inhibits their ability to interact with cyclins D and to phosphorylate the retinoblastoma protein.
Uniprot:
P51480
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Cyclin-dependent kinase inhibitor 2A, isoforms 1/2
Sub Unit:
Heterodimer with CDK4 or CDK6. Predominamt P16 complexes contained CDK6. Interacts with CDK4 (both 'T-172'-phosphorylated and non-phosphorylated forms); the interaction inhibits cyclin D-CDK4 kinase activity. Interacts with ISCO2.
Research Area:
Cancer
Subcellular Location:
Cytoplasm Nucleus
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
p16-INK4A: a cell-cycle regulatory protein that interacts with CDK4 and CDK6, inhibiting their ability to interact with cyclins D. Inhibits the phosphorylation of the retinoblastoma protein by CDK4 or CDK6, and entry into the S phase of the cell cycle. The p16INK4A and p14ARF proteins are encoded by CDKN2A, a known tumour suppressor gene in multiple cancers. CDKN2A is inactivated in 72% of cases of lung squamous cell carcinoma: 21% by epigenetic silencing by methylation, 18% inactivating mutation, 4% by exon 1b skipping, and 29% by homozygous deletion. Defects in CDKN2A are the cause of familial atypical multiple mole melanoma-pancreatic carcinoma syndrome, Li-Fraumeni syndrome, and the melanoma-astrocytoma syndrome. The melanoma-astrocytoma syndrome is characterized by a dual predisposition to melanoma and neural system tumors, commonly astrocytoma. Four alternatively spliced p16 isoforms have been reported. Two alternatively spliced isoforms of ARF have been reported.Protein type: Cell cycle regulation; Nucleolus; Tumor suppressorChromosomal Location of Human Ortholog: 4 C4|4 42.15 cMCellular Component: cytoplasm; granular component; mitochondrion; nuclear body; nucleolus; nucleoplasm; nucleus; protein complex; senescence-associated heterochromatin focusMolecular Function: cyclin-dependent protein serine/threonine kinase inhibitor activity; DNA binding; DNA binding transcription factor activity; NF-kappaB binding; p53 binding; protein binding; protein kinase binding; protein N-terminus binding; RNA binding; SUMO transferase activity; transcription factor binding; ubiquitin ligase inhibitor activity; ubiquitin-protein transferase inhibitor activityBiological Process: activation of cysteine-type endopeptidase activity involved in apoptotic process; aging; amyloid fibril formation; apoptosis; apoptotic mitochondrial changes; cell aging; cell cycle; cell cycle arrest; cellular response to hydrogen peroxide; cellular senescence; epidermis development; G1/S transition of mitotic cell cycle; glucose homeostasis; mitochondrial depolarization; mitochondrion degradation; negative regulation of B cell proliferation; negative regulation of cell cycle; negative regulation of cell growth; negative regulation of cell proliferation; negative regulation of cell-matrix adhesion; negative regulation of cyclin-dependent protein serine/threonine kinase activity; negative regulation of hepatocyte proliferation; negative regulation of immature T cell proliferation in the thymus; negative regulation of mammary gland epithelial cell proliferation; negative regulation of NF-kappaB transcription factor activity; negative regulation of phosphorylation; negative regulation of protein binding; negative regulation of protein kinase activity; negative regulation of protein neddylation; negative regulation of protein ubiquitination involved in ubiquitin-dependent protein catabolic process; negative regulation of transcription, DNA-dependent; negative regulation of ubiquitin protein ligase activity; negative regulation of ubiquitin-protein ligase activity; positive regulation of apoptosis; positive regulation of apoptotic process involved in mammary gland involution; positive regulation of cell cycle arrest; positive regulation of cellular senescence; positive regulation of DNA damage response, signal transduction by p53 class mediator; positive regulation of gene expression; positive regulation of macrophage apoptotic process; positive regulation of protein localization to nucleus; positive regulation of protein sumoylation; positive regulation of smooth muscle cell apoptotic process; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-templated; protein destabilization; protein polyubiquitination; protein stabilization; regulation of cyclin-dependent protein kinase activity; regulation of G2/M transition of mitotic cell cycle; regulation of gene expression; regulation of nucleocytoplasmic transport; regulation of protein export from nucleus; regulation of protein stability; regulation of transcription, DNA-templated; replicative senescence; response to drug; response to organic cyclic compound; response to organonitrogen compound; rRNA processing; rRNA transcription; senescence-associated heterochromatin focus assembly; somatic stem cell division; somatic stem cell maintenance; transcription, DNA-dependent
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
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.
