The Bovine Cyclin Dependent Kinase 4 (CDK4) ELISA Kit from AssayGenie is a powerful tool for the accurate quantification of CDK4 levels in bovine samples. This kit offers excellent sensitivity and specificity, ensuring precise and reliable results for various research applications.CDK4 is a key regulator of cell cycle progression, playing a critical role in cell division and growth. Dysregulation of CDK4 has been linked to various diseases, including cancer, making it an important biomarker for studying disease mechanisms and potential therapeutic targets.
With its high-quality antibodies and optimized assay protocols, the AssayGenie Bovine CDK4 ELISA Kit provides researchers with a valuable tool for investigating CDK4 levels in bovine samples. Whether studying cell cycle regulation, cancer biology, or other related fields, this ELISA kit offers a comprehensive solution for accurate and efficient protein quantification.
Product Name:
Bovine Cyclin-dependent kinase 4 (CDK4) ELISA Kit
SKU:
BOEB0561
Size:
96T
Target:
Bovine Cyclin-dependent kinase 4 (CDK4)
Synonyms:
Cell division protein kinase 4
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Bovine
Detection Range:
0.156-10ng/mL
Sensitivity:
0.059ng/mL
Intra CV:
Provided with the Kit
Inter CV:
Provided with the Kit
Linearity:
Provided with the Kit
Recovery:
Provided with the Kit
Function:
Ser/Thr-kinase component of cyclin D-CDK4 (DC) complexes that phosphorylate and inhibit members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complexes and the subsequent transcription of E2F target genes which are responsible for the progression through the G(1) phase. Hypophosphorylates RB1 in early G(1) phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also phosphorylates SMAD3 in a cell-cycle-dependent manner and represses its transcriptional activity. Component of the ternary complex, cyclin D/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex.
Uniprot:
Q32KY4
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant bovine Cyclin-dependent kinase 4
Sub Unit:
Component of the D-CDK4 complex, composed of CDK4 and some D-type G1 cyclin (CCND1, CCND2 or CCND3). Interacts directly in the complex with CCND1, CCND2 or CCND3. Interacts with SEI1 and ZNF655. Forms a ternary complex, cyclin D-CDK4-CDKN1B, involved in modulating CDK4 enzymatic activity. Interacts directly with CDKN1B (phosphorylated on 'Tyr-88' and 'Tyr-89'); the interaction allows assembly of the cyclin D-CDK4 complex, Thr-172 phosphorylation, nuclear translocation and enhances the cyclin D-CDK4 complex activity. CDK4 activity is either inhibited or enhanced depending on stoichiometry of complex. The non-tyrosine-phosphorylated form of CDKN1B prevents T-loop phosphorylation of CDK4 producing inactive CDK4. Interacts (unphosphorylated form) with CDK2. Also forms ternary complexes with CDKN1A or CDKN2A. Interacts directly with CDKN1A (via its N-terminal); the interaction promotes the assembly of the cyclin D-CDK4 complex, its nuclear translocation and promotes the cyclin D-dependent enzyme activity of CDK4. Interacts with CCND1; the interaction is prevented with the binding of CCND1 to INSM1 during cell cycle progression. Interacts with CEBPA (when phosphorylated). Interacts with FNIP1 and FNIP2.
Research Area:
Cancer
Subcellular Location:
Cytoplasm Nucleus Membrane Cytoplasmic when non-complexed. Forms a cyclin D-CDK4 complex in the cytoplasm as cells progress through G(1) phase. The complex accumulates on the nuclear membrane and enters the nucleus on transition from G(1) to S phase. Also present in nucleoli and heterochromatin lumps. Colocalizes with RB1 after release into the nucleus (By similarity).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
Function: Ser/Thr-kinase component of cyclin D-CDK4 (DC) complexes that phosphorylate and inhibit members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G1/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complexes and the subsequent transcription of E2F target genes which are responsible for the progression through the G1 phase. Hypophosphorylates RB1 in early G1 phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also phosphorylates SMAD3 in a cell-cycle-dependent manner and represses its transcriptional activity. Component of the ternary complex, cyclin D/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex Catalytic activity: ATP + a protein = ADP + a phosphoprotein.Enzyme regulation: Both phosphorylation at Thr-172 and binding of a D-type cyclin are necessary for enzymatic activity. Full activation of the cyclin-D-CDK4 complex appears to require other factors such as recruitment of the substrate via a substrate recruitment motif, and/or formation of the CDKN1B ternary complex. Inhibited by INK4 family members. In resting cells, the non-tyrosine-phosphorylated form of CDKN1B prevents phosphorylation at Thr-172 and inactivation, while, in proliferating cells, tyrosine phosphorylation of CDKN1B allows phosphorylation of Thr-172 of CDK4 and subsequennt activation.
cyclin-dependent kinase 4; cell division protein kinase 4
UniProt Protein Name:
Cyclin-dependent kinase 4
UniProt Synonym Protein Names:
Cell division protein kinase 4
Protein Family:
Cyclin-dependent kinase
UniProt Gene Name:
CDK4
UniProt Entry Name:
CDK4_BOVIN
Component
Quantity (96 Assays)
Storage
ELISA Microplate (Dismountable)
8×12 strips
-20°C
Lyophilized Standard
2
-20°C
Sample Diluent
20ml
-20°C
Assay Diluent A
10mL
-20°C
Assay Diluent B
10mL
-20°C
Detection Reagent A
120µL
-20°C
Detection Reagent B
120µL
-20°C
Wash Buffer
30mL
4°C
Substrate
10mL
4°C
Stop Solution
10mL
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
*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.
