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Rat Proliferating cell nuclear antigen (Pcna) ELISA Kit (RTEB0361)

SKU:
RTEB0361
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P04961
ELISA Type:
Sandwich
Synonyms:
PCNA
Reactivity:
Rat
€649
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Description

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Rat Proliferating cell nuclear antigen (Pcna) ELISA Kit

The Rat Proliferating Cell Nuclear Antigen (PCNA) ELISA Kit is specifically designed for the accurate measurement of PCNA levels in rat serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit provides reliable and reproducible results, making it an essential tool for various research applications.PCNA is a key protein involved in DNA replication and repair, serving as a marker for cell proliferation. Elevated PCNA levels have been associated with cancer and other proliferative disorders, making it a valuable biomarker for studying these conditions and developing novel therapies.

This ELISA kit offers researchers a precise and efficient way to analyze PCNA levels in rat samples, providing valuable insights into cell proliferation and potential disease mechanisms. With its user-friendly format and accurate results, the Rat PCNA ELISA Kit is a valuable addition to any laboratory focusing on cell biology and disease research.

Product Name:Rat Proliferating cell nuclear antigen (Pcna) ELISA Kit
SKU:RTEB0361
Size:96T
Target:Rat Proliferating cell nuclear antigen (Pcna)
Synonyms:Cyclin, PCNA
Assay Type:Sandwich
Detection Method:ELISA
Reactivity:Rat
Detection Range:0.156-10ng/mL
Sensitivity:0.085ng/mL
Intra CV:Provided with the Kit
Inter CV:Provided with the Kit
Linearity:Provided with the Kit
Recovery:Provided with the Kit
Function:Auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand. Induces a robust stimulatory effect on the 3'-5' exonuclease and 3'-phosphodiesterase, but not apurinic-apyrimidinic (AP) endonuclease, APEX2 activities. Has to be loaded onto DNA in order to be able to stimulate APEX2. Plays a key role in DNA damage response (DDR) by being conveniently positioned at the replication fork to coordinate DNA replication with DNA repair and DNA damage tolerance pathways. Acts as a loading platform to recruit DDR proteins that allow completion of DNA replication after DNA damage and promote postreplication repair: Monoubiquitinated PCNA leads to recruitment of translesion (TLS) polymerases, while 'Lys-63'-linked polyubiquitination of PCNA is involved in error-free pathway and employs recombination mechanisms to synthesize across the lesion.
Uniprot:P04961
Sample Type:Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:Natural and recombinant rat Proliferating cell nuclear antigen
Sub Unit:Homotrimer. Interacts with p300/EP300; the interaction occurs on chromatin in UV-irradiated damaged cells. Interacts with CREBBP (via transactivation domain and C-terminus); the interaction occurs on chromatin in UV-irradiated damaged cells. Directly interacts with POLD1, POLD3 and POLD4 subunits of the DNA polymerase delta complex, POLD3 being the major interacting partner; the interaction with POLD3 is inhibited by CDKN1A/p21(CIP1). Forms a complex with activator 1 heteropentamer in the presence of ATP. Interacts with EXO1, POLH, POLK, DNMT1, ERCC5, FEN1, CDC6 and POLDIP2. Interacts with APEX2; this interaction is triggered by reactive oxygen species and increased by misincorporation of uracil in nuclear DNA. Forms a ternary complex with DNTTIP2 and core histone (By similarity). Interacts with KCTD10 (PubMed:15982757). Interacts with PPP1R15A (By similarity). Directly interacts with BAZ1B. Interacts with HLTF and SHPRH. Interacts with NUDT15. Interaction is disrupted in response to UV irradiation and acetylation. Interacts with CDKN1A/p21(CIP1) and CDT1; interacts via their PIP-box which also recruits the DCX(DTL) complex. The interaction with CDKN1A inhibits POLD3 binding. Interacts with DDX11. Interacts with EGFR; positively regulates PCNA. Interacts with PARPBP. Interacts (when ubiquitinated) with SPRTN; leading to enhance RAD18-mediated PCNA ubiquitination. Interacts (when polyubiquitinated) with ZRANB3. Interacts with SMARCAD1. Interacts with CDKN1C. Interacts with PCLAF (via PIP-box). Interacts with RTEL1 (via PIP-box); the interaction is direct and essential for the suppression of telomere fragility. Interacts with FAM111A (via PIP-box); the interaction is direct and required for PCNA loading on chromatin binding. Interacts with LIG1. Interacts with SETMAR. Interacts with ANKRD17. Interacts with FBXO18/FBH1 (via PIP-box); the interaction recruits the DCX(DTL) complex and promotes ubiquitination and degradation of FBXO18/FBH1. Interacts with POLN.
Research Area:Cancer
Subcellular Location:Nucleus Forms nuclear foci representing sites of ongoing DNA replication and vary in morphology and number during S phase. Together with APEX2, is redistributed in discrete nuclear foci in presence of oxidative DNA damaging agents. Colocalizes with CREBBP, EP300 and POLD1 to sites of DNA damage (By similarity).
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:PCNA: This protein is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand. Induces a robust stimulatory effect on the 3'- 5' exonuclease and 3'-phosphodiesterase, but not apurinic- apyrimidinic (AP) endonuclease, APEX2 activities. Has to be loaded onto DNA in order to be able to stimulate APEX2. Homotrimer. Forms a complex with activator 1 heteropentamer in the presence of ATP. Interacts with EXO1, POLH, POLK, DNMT1, ERCC5, FEN1, CDC6 and POLDIP2. Interacts with APEX2; this interaction is triggered by reactive oxygen species and increased by misincorporation of uracil in nuclear DNA. Forms a ternary complex with DNTTIP2 and core histone. Interacts with KCTD10 and PPP1R15A. Interacts with POLD1, POLD3 and POLD4. Interacts with BAZ1B; the interaction is direct. Interacts with HLTF and SHPRH. Interacts with NUDT15. Interaction is disrupted in response to UV irradiation and acetylation. Interacts with p21Cip1/p21(CIP1) and CDT1; interacts via their PIP-box which also recruits the DCX(DTL) complex. Interacts with DDX11. Interacts with EGFR; positively regulates PCNA. Interacts with C12orf48/PARI. Interacts with SMARCAD1. Belongs to the PCNA family.
UniProt Protein Details:

Protein type:DNA replication; Cell cycle regulation

Cellular Component: cell; centrosome; cyclin-dependent protein kinase holoenzyme complex; cytoplasm; intracellular; nuclear chromosome, telomeric region; nuclear lamina; nuclear replication fork; nucleoplasm; nucleus; PCNA complex; replication fork

Molecular Function:chromatin binding; damaged DNA binding; dinucleotide insertion or deletion binding; DNA polymerase processivity factor activity; enzyme binding; estrogen receptor binding; histone acetyltransferase binding; identical protein binding; MutLalpha complex binding; protein binding; purine-specific mismatch base pair DNA N-glycosylase activity; receptor tyrosine kinase binding; transcription factor binding

Biological Process: base-excision repair, gap-filling; bypass DNA synthesis; epithelial cell differentiation; heart development; leading strand elongation; mismatch repair; negative regulation of transcription from RNA polymerase II promoter; positive regulation of deoxyribonuclease activity; positive regulation of DNA repair; positive regulation of DNA replication; replication fork processing; response to cadmium ion; response to estradiol stimulus; response to lipid; response to oxidative stress

NCBI Summary:cell cycle protein that may contribute to the repair of DNA damage and to cell proliferation caused by exposure to pulmonary toxicants; may influence cell survival in ischaemically compromised tissue [RGD, Feb 2006]
UniProt Code:P04961
NCBI GenInfo Identifier:129698
NCBI Gene ID:25737
NCBI Accession:P04961.1
UniProt Related Accession:P04961
Molecular Weight:28,749 Da
NCBI Full Name:Proliferating cell nuclear antigen
NCBI Synonym Full Names:proliferating cell nuclear antigen
NCBI Official Symbol:Pcna
NCBI Official Synonym Symbols:PCNAR; Pcna/cyclin
NCBI Protein Information:proliferating cell nuclear antigen
UniProt Protein Name:Proliferating cell nuclear antigen
UniProt Synonym Protein Names:Cyclin
Protein Family:PCNA-associated factor
UniProt Gene Name:Pcna
UniProt Entry Name:PCNA_RAT
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.

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