Description
SENP5 Colorimetric Cell-Based ELISA Kit
The SENP5 Colorimetric Cell-Based ELISA Kit from AssayGenie is a powerful tool for researchers looking to study SENP5 levels in cell culture supernatants. SENP5 is a key regulator of the SUMOylation process, influencing a variety of cellular pathways and functions.This kit offers high sensitivity and specificity, ensuring accurate and reproducible results, making it perfect for a wide range of cell biology and molecular biology research applications. By measuring SENP5 levels, researchers can gain valuable insights into the role of SUMOylation in various cellular processes, including DNA repair, transcriptional regulation, and protein stability.
Whether studying the impact of SENP5 on cell proliferation, apoptosis, or gene expression, the SENP5 Colorimetric Cell-Based ELISA Kit provides a reliable and efficient method for quantifying SENP5 levels in cell samples. Invest in this kit today and unlock new possibilities for your research.
| Product Name: | SENP5 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00852 |
| ELISA Type: | Cell-Based |
| Target: | SENP5 |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The SENP5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SENP5 protein expression profile in cells. The kit can be used for measuring the relative amounts of SENP5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on SENP5.
Qualitative determination of SENP5 concentration is achieved by an indirect ELISA format. In essence, SENP5 is captured by SENP5-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: 205564, UniProt ID: Q96HI0, OMIM: 612845, Unigene: Hs.240770 |
| Gene Symbol: | SENP5 |
| Sub Type: | None |
| UniProt Protein Function: | SENP5: Protease that catalyzes two essential functions in the SUMO pathway: processing of full-length SUMO3 to its mature form and deconjugation of SUMO2 and SUMO3 from targeted proteins. Has weak proteolytic activity against full-length SUMO1 or SUMO1 conjugates. Required for cell division. Belongs to the peptidase C48 family. |
| UniProt Protein Details: | Protein type:Nucleolus; Protease; EC 3.4.22.68 Chromosomal Location of Human Ortholog: 3q29 Cellular Component: intracellular membrane-bound organelle; nucleolus; nucleoplasm; nucleus Molecular Function:endopeptidase activity; protein binding; SUMO-specific protease activity Biological Process: protein desumoylation; protein sumoylation |
| NCBI Summary: | The reversible posttranslational modification of proteins by the addition of small ubiquitin-like SUMO proteins (see SUMO1; MIM 601912) is required for numerous biologic processes. SUMO-specific proteases, such as SENP5, are responsible for the initial processing of SUMO precursors to generate a C-terminal diglycine motif required for the conjugation reaction. They also have isopeptidase activity for the removal of SUMO from high molecular mass SUMO conjugates (Di Bacco et al., 2006 [PubMed 16738315]).[supplied by OMIM, Jun 2009] |
| UniProt Code: | Q96HI0 |
| NCBI GenInfo Identifier: | 296452962 |
| NCBI Gene ID: | 205564 |
| NCBI Accession: | Q96HI0.3 |
| UniProt Secondary Accession: | Q96HI0,Q96SA5, B4DY82, |
| UniProt Related Accession: | Q96HI0 |
| Molecular Weight: | 81,310 Da |
| NCBI Full Name: | Sentrin-specific protease 5 |
| NCBI Synonym Full Names: | SUMO1/sentrin specific peptidase 5 |
| NCBI Official Symbol: | SENP5 |
| NCBI Protein Information: | sentrin-specific protease 5 |
| UniProt Protein Name: | Sentrin-specific protease 5 |
| UniProt Synonym Protein Names: | Sentrin/SUMO-specific protease SENP5 |
| Protein Family: | Sentrin-specific protease |
| UniProt Gene Name: | SENP5 |
| UniProt Entry Name: | SENP5_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-SENP5 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.)
