Description
| Product Name: | PML Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00826 |
| ELISA Type: | Cell-Based |
| Target: | PML |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The PML Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PML protein expression profile in cells. The kit can be used for measuring the relative amounts of PML in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PML.
Qualitative determination of PML concentration is achieved by an indirect ELISA format. In essence, PML is captured by PML-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 5371, UniProt ID: P29590, OMIM: 102578, Unigene: Hs.526464 |
| Gene Symbol: | PML |
| Sub Type: | None |
| UniProt Protein Function: | PML: a zinc-finger protein that can regulate transcription and can localize to nuclear bodies. Cytoplasmic forms regulate glycolysis by inhibiting the tetrameric form of PKM2. Together with SATB1, involved in local chromatin-loop remodeling and gene expression regulation at the MHC-I locus. Interacts with SIRT1, TOPBP1, TRIM27 and TRIM69. Sumoylated forms interact with SATB1 and localize to the PML nuclear bodies. Sumoylation on three sites is required for nuclear body formation. Sumoylation on Lys-160 is a prerequisite for sumoylation on Lys-65. The B1 box and the RING finger are also required for localization to nuclear bodies. May play an important role in recruitment of ELF4 into PML nuclear bodies. A chromosomal aberration involving PML can cause acute promyelocytic leukemia (APL). Seven alternatively-spliced human isoforms have been reported. |
| UniProt Protein Details: | Protein type:Ubiquitin conjugating system; Tumor suppressor; Transcription factor; Nucleolus; Oncoprotein Chromosomal Location of Human Ortholog: 15q22 Cellular Component: nucleoplasm; PML body; nuclear membrane; nuclear matrix; early endosome membrane; cytoplasm; nucleolus; nucleus; cytosol Molecular Function:protein binding; protein homodimerization activity; SUMO binding; DNA binding; zinc ion binding; protein heterodimerization activity; ubiquitin protein ligase binding; transcription coactivator activity; SMAD binding; cobalt ion binding Biological Process: retinoic acid receptor signaling pathway; proteasomal ubiquitin-dependent protein catabolic process; viral reproduction; apoptosis; positive regulation of apoptosis involved in mammary gland involution; regulation of MHC class I biosynthetic process; PML body organization and biogenesis; SMAD protein nuclear translocation; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; entrainment of circadian clock by photoperiod; negative regulation of cell proliferation; positive regulation of histone deacetylation; regulation of transcription, DNA-dependent; transforming growth factor beta receptor signaling pathway; response to gamma radiation; protein complex assembly; negative regulation of mitotic cell cycle; circadian regulation of gene expression; maintenance of protein localization in nucleus; cell cycle arrest; defense response to virus; negative regulation of telomere maintenance via telomerase; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; response to UV; caspase activation; regulation of protein amino acid phosphorylation; cell fate commitment; protein stabilization; transcription, DNA-dependent; cytokine and chemokine mediated signaling pathway; common-partner SMAD protein phosphorylation; regulation of circadian rhythm; negative regulation of telomerase activity; endoplasmic reticulum calcium ion homeostasis; negative regulation of angiogenesis; DNA damage response, signal transduction resulting in induction of apoptosis; response to cytokine stimulus; response to hypoxia; innate immune response; myeloid cell differentiation; negative regulation of translation in response to oxidative stress; negative regulation of cell growth; positive regulation of defense response to virus by host; negative regulation of transcription, DNA-dependent; induction of apoptosis by oxidative stress; protein targeting |
| NCBI Summary: | The protein encoded by this gene is a member of the tripartite motif (TRIM) family. The TRIM motif includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled-coil region. This phosphoprotein localizes to nuclear bodies where it functions as a transcription factor and tumor suppressor. Its expression is cell-cycle related and it regulates the p53 response to oncogenic signals. The gene is often involved in the translocation with the retinoic acid receptor alpha gene associated with acute promyelocytic leukemia (APL). Extensive alternative splicing of this gene results in several variations of the protein's central and C-terminal regions; all variants encode the same N-terminus. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P29590 |
| NCBI GenInfo Identifier: | 15451765 |
| NCBI Gene ID: | 5371 |
| NCBI Accession: | NP_150242 |
| UniProt Related Accession: | P29590 |
| Molecular Weight: | 97.516 |
| NCBI Full Name: | protein PML isoform 9 |
| NCBI Synonym Full Names: | PML nuclear body scaffold |
| NCBI Official Symbol: | PML |
| NCBI Official Synonym Symbols: | MYL; RNF71; PP8675; TRIM19 |
| NCBI Protein Information: | protein PML |
| UniProt Protein Name: | Protein PML |
| UniProt Synonym Protein Names: | Promyelocytic leukemia protein; RING finger protein 71; Tripartite motif-containing protein 19 |
| Protein Family: | Protein |
| UniProt Gene Name: | PML |
| UniProt Entry Name: | PML_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies (Anti-PML Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
Additional Information
Product Type: |
ELISA Kit |
ELISA Type: |
Cell Based |
Research Area: |
Immunology |
Reactivity: |
Human |
Detection Method: |
Colorimetric |
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