Mouse Cell Biology ELISA Kits 2
Mouse Proliferating cell nuclear antigen (Pcna) ELISA Kit
- SKU:
- MOEB0476
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P17918
- Range:
- 0.156-10 ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- Pcna, PCNA, Cyclin
- Reactivity:
- Mouse
Description
| Product Name: | Mouse Proliferating cell nuclear antigen (Pcna) ELISA Kit |
| Product Code: | MOEB0476 |
| Alias: | Proliferating cell nuclear antigen, PCNA, Cyclin, Pcna |
| Uniprot: | P17918 |
| Reactivity: | Mouse |
| Range: | 0.156-10 ng/mL |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| 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:Cell cycle regulation; DNA replication Cellular Component: nucleoplasm; nuclear lamina; centrosome; PCNA complex; cytoplasm; nuclear replication fork; cyclin-dependent protein kinase holoenzyme complex; replication fork; intracellular; nucleus Molecular Function:identical protein binding; protein binding; DNA polymerase processivity factor activity; DNA binding; purine-specific mismatch base pair DNA N-glycosylase activity; estrogen receptor binding; MutLalpha complex binding; dinucleotide insertion or deletion binding; receptor tyrosine kinase binding; transcription factor binding Biological Process: base-excision repair, gap-filling; bypass DNA synthesis; mismatch repair; regulation of catalytic activity; positive regulation of deoxyribonuclease activity; negative regulation of transcription from RNA polymerase II promoter; DNA repair; response to lipid; DNA replication; leading strand elongation; response to DNA damage stimulus; regulation of DNA replication |
| UniProt Code: | P17918 |
| NCBI GenInfo Identifier: | 7242171 |
| NCBI Gene ID: | 18538 |
| NCBI Accession: | NP_035175.1 |
| UniProt Related Accession: | P17918 |
| Molecular Weight: | 28,785 Da |
| NCBI Full Name: | proliferating cell nuclear antigen |
| NCBI Synonym Full Names: | proliferating cell nuclear antigen |
| NCBI Official Symbol: | Pcna |
| NCBI Protein Information: | proliferating cell nuclear antigen; cyclin |
| UniProt Protein Name: | Proliferating cell nuclear antigen |
| UniProt Synonym Protein Names: | Cyclin |
| Protein Family: | Proliferating cell nuclear antigen |
| UniProt Gene Name: | Pcna |
| UniProt Entry Name: | PCNA_MOUSE |
| 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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