Description
TSN Colorimetric Cell-Based ELISA
The TSN Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for researchers looking to study angiogenin levels in cell culture supernatants. This kit offers unparalleled sensitivity and specificity, making it the perfect choice for accurate and reproducible results in a variety of research applications.Angiogenin is a key player in angiogenesis, the process of forming new blood vessels, and has been implicated in conditions such as cancer, cardiovascular diseases, and neurodegenerative disorders.
By accurately measuring angiogenin levels, researchers can gain valuable insights into these conditions and potentially develop new therapeutic strategies.The TSN Colorimetric Cell-Based ELISA Kit is a reliable and easy-to-use solution for studying angiogenin levels in cell cultures, providing researchers with the tools they need to advance their research and contribute to the understanding of angiogenesis and its role in disease progression.
| Product Name: | TSN Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01110 |
| ELISA Type: | Cell-Based |
| Target: | TSN |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The TSN Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TSN protein expression profile in cells. The kit can be used for measuring the relative amounts of TSN in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on TSN.
Qualitative determination of TSN concentration is achieved by an indirect ELISA format. In essence, TSN is captured by TSN-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: 7247, UniProt ID: Q15631, OMIM: 600575, Unigene: Hs.75066 |
| Gene Symbol: | TSN |
| Sub Type: | None |
| UniProt Protein Function: | TSN: DNA-binding protein that specifically recognizes consensus sequences at the breakpoint junctions in chromosomal translocations, mostly involving immunoglobulin (Ig)/T-cell receptor gene segments. Seems to recognize single-stranded DNA ends generated by staggered breaks occurring at recombination hot spots. Belongs to the translin family. |
| UniProt Protein Details: | Protein type:DNA-binding; Nuclear receptor co-regulator; EC 3.1.-.- Chromosomal Location of Human Ortholog: 2q21.1 Cellular Component: cytosol Molecular Function:DNA binding; protein binding Biological Process: DNA recombination; RNA-mediated gene silencing |
| NCBI Summary: | This gene encodes a DNA-binding protein which specifically recognizes conserved target sequences at the breakpoint junction of chromosomal translocations. Translin polypeptides form a multimeric structure that is responsible for its DNA-binding activity. Recombination-associated motifs and translin-binding sites are present at recombination hotspots and may serve as indicators of breakpoints in genes which are fused by translocations. These binding activities may play a crucial role in chromosomal translocation in lymphoid neoplasms. This protein encoded by this gene, when complexed with translin-associated protein X, also forms a Mg ion-dependent endoribonuclease that promotes RNA-induced silencing complex (RISC) activation. Alternative splicing results in multiple transcript variants. [provided by RefSeq, May 2012] |
| UniProt Code: | Q15631 |
| NCBI GenInfo Identifier: | 6136060 |
| NCBI Gene ID: | 7247 |
| NCBI Accession: | Q15631.1 |
| UniProt Secondary Accession: | Q15631,Q5U0K7, B7Z3X8, |
| UniProt Related Accession: | Q15631 |
| Molecular Weight: | 17,279 Da |
| NCBI Full Name: | Translin |
| NCBI Synonym Full Names: | translin |
| NCBI Official Symbol: | TSN |
| NCBI Official Synonym Symbols: | C3PO; RCHF1; TBRBP; TRSLN; BCLF-1; REHF-1 |
| NCBI Protein Information: | translin |
| UniProt Protein Name: | Translin |
| UniProt Synonym Protein Names: | Component 3 of promoter of RISC; C3PO |
| Protein Family: | Translin |
| UniProt Gene Name: | TSN |
| UniProt Entry Name: | TSN_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-TSN 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.)
