The Porcine Cellular Tumor Antigen p53 (TP53) ELISA Kit is a reliable and accurate tool for detecting levels of TP53 in porcine samples, including serum, plasma, and cell culture supernatants. With high sensitivity and specificity, this kit delivers precise and reproducible results, making it an ideal choice for a variety of research applications.TP53, also known as the cellular tumor antigen p53, is a key protein involved in regulating cell growth and division, as well as DNA repair and apoptosis.
Mutations in TP53 have been associated with various types of cancer, making it a crucial biomarker for studying tumorigenesis and potential therapeutic interventions.With the Porcine Cellular Tumor Antigen p53 (TP53) ELISA Kit, researchers can accurately measure TP53 levels in porcine samples, providing valuable insights into the role of this protein in cancer development and progression.
Acts as a tumor suppressor in many tumor types; induces growth arrest or apoptosis depending on the physiological circumstances and cell type. Involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. One of the activated genes is an inhibitor of cyclin-dependent kinases. Apoptosis induction seems to be mediated either by stimulation of BAX and FAS antigen expression, or by repression of Bcl-2 expression. In cooperation with mitochondrial PPIF is involved in activating oxidative stress-induced necrosis; the function is largely independent of transcription. Prevents CDK7 kinase activity when associated to CAK complex in response to DNA damage, thus stopping cell cycle progression. Induces the transcription of long intergenic non-coding RNA p21 (lincRNA-p21) and lincRNA-Mkln1. LincRNA-p21 participates in TP53-dependent transcriptional repression leading to apoptosis and seem to have to effect on cell-cycle regulation. Regulates the circadian clock by repressing CLOCK-ARNTL/BMAL1-mediated transcriptional activation of PER2.
Uniprot:
Q9TUB2
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant pig Cellular tumor antigen p53
Sub Unit:
Binds DNA as a homotetramer. Found in a complex with CABLES1 and TP73. Interacts with histone acetyltransferases EP300 and methyltransferases HRMT1L2 and CARM1, and recruits them to promoters. The C-terminus interacts with TAF1, when TAF1 is part of the TFIID complex. Interacts with HIPK1, HIPK2, AXIN1, and TP53INP1. Part of a complex consisting of TP53, HIPK2 and AXIN1. Interacts with WWOX. Interacts with USP7 and SYVN1. Interacts with HSP90AB1. Interacts with ARMD10, BANP, CDKN2AIP, NUAK1, STK11/LKB1, UHRF2 and E4F1. Interacts with CHD8, leading to recruit histone H1 and prevent transactivation activity. Interacts with YWHAZ; the interaction enhances TP53 transcriptional activity. Phosphorylation of YWHAZ on 'Ser-58' inhibits this interaction. Directly interacts with FBXO42; leading to ubiquitination and degradation of TP53. Interacts with AURKA, DAXX, BRD7 and TRIM24. Interacts (when monomethylated at Lys-375) with L3MBTL1. Interacts with GRK5. Binds to the CAK complex (CDK7, cyclin H and MAT1) in response to DNA damage. Interacts with CDK5 in neurons. Interacts with AURKB, SETD2, UHRF2 and NOC2L. Interacts (via N-terminus) with PTK2/FAK1; this promotes ubiquitination by MDM2. Interacts with PTK2B/PYK2; this promotes ubiquitination by MDM2. Interacts with PRKCG. Interacts with PPIF; the association implicates preferentially tetrameric TP53, is induced by oxidative stress and is impaired by cyclosporin A (CsA). Interacts with SNAI1; the interaction induces SNAI1 degradation via MDM2-mediated ubiquitination and inhibits SNAI1-induced cell invasion. Interacts with KAT6A. Interacts with UBC9. Forms a complex with UBC9 and PRKRA. Interacts with ZNF385B; the interaction is direct. Interacts (via DNA-binding domain) with ZNF385A; the interaction is direct and enhances p53/TP53 transactivation functions on cell-cycle arrest target genes, resulting in growth arrest. Interacts with ANKRD2. Interacts with RFFL and RNF34; involved in p53/TP53 ubiquitination. Interacts with MTA1 and RFWD2. Interacts with CCAR2 (via N-terminus). Interacts with MORC3. Interacts (via C-terminus) with POU4F2 (via C-terminus).
Research Area:
Neurosciences
Subcellular Location:
Cytoplasm Nucleus Endoplasmic reticulum Mitochondrion matrix Interaction with BANP promotes nuclear localization. Translocates to mitochondria upon oxidative stress (By similarity).
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
Function: Acts as a tumor suppressor in many tumor types; induces growth arrest or apoptosis depending on the physiological circumstances and cell type. Involved in cell cycle regulation as a trans-activator that acts to negatively regulate cell division by controlling a set of genes required for this process. One of the activated genes is an inhibitor of cyclin-dependent kinases. Apoptosis induction seems to be mediated either by stimulation of BAX and FAS antigen expression, or by repression of Bcl-2 expression. In cooperation with mitochondrial PPIF is involved in activating oxidative stress-induced necrosis; the function is largely independent of transcription. Prevents CDK7 kinase activity when associated to CAK complex in response to DNA damage, thus stopping cell cycle progression. Induces the transcription of long intergenic non-coding RNA p21 (lincRNA-p21) and lincRNA-Mkln1. LincRNA-p21 participates in TP53-dependent transcriptional repression leading to apoptosis and seem to have to effect on cell-cycle regulation Cofactor: Binds 1 zinc ion per subunit
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.
