Prostate-specific Antigen Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB00832
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Reactivity:
- Human
- Detection Method:
- Colorimetric
Description
| Product Name: | Prostate-specific Antigen Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00832 |
| ELISA Type: | Cell-Based |
| Target: | Prostate-specific Antigen |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Prostate-specific Antigen Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Prostate-specific Antigen protein expression profile in cells. The kit can be used for measuring the relative amounts of Prostate-specific Antigen in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Prostate-specific Antigen.
Qualitative determination of Prostate-specific Antigen concentration is achieved by an indirect ELISA format. In essence, Prostate-specific Antigen is captured by Prostate-specific Antigen-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: 354, UniProt ID: P07288, OMIM: 176820, Unigene: Hs.171995 |
| Gene Symbol: | KLK3 |
| Sub Type: | None |
| UniProt Protein Function: | PSA: Hydrolyzes semenogelin-1 thus leading to the liquefaction of the seminal coagulum. Forms heterodimer with SERPINA5. Inhibited by SERPINA5. Activity is strongly inhibited by Zn2+, 100 times more abundant in semen than in serum. This inhibition is relieved by exposure to semenogelins, which are avid zinc binders. Belongs to the peptidase S1 family. Kallikrein subfamily. |
| UniProt Protein Details: | Protein type:Protease; Secreted, signal peptide; EC 3.4.21.77; Secreted Chromosomal Location of Human Ortholog: 19q13.41 Cellular Component: extracellular region; nucleus Molecular Function:protein binding; serine-type peptidase activity; serine-type endopeptidase activity Biological Process: negative regulation of angiogenesis; cellular protein metabolic process; proteolysis |
| NCBI Summary: | Kallikreins are a subgroup of serine proteases having diverse physiological functions. Growing evidence suggests that many kallikreins are implicated in carcinogenesis and some have potential as novel cancer and other disease biomarkers. This gene is one of the fifteen kallikrein subfamily members located in a cluster on chromosome 19. Its protein product is a protease present in seminal plasma. It is thought to function normally in the liquefaction of seminal coagulum, presumably by hydrolysis of the high molecular mass seminal vesicle protein. Serum level of this protein, called PSA in the clinical setting, is useful in the diagnosis and monitoring of prostatic carcinoma. Alternate splicing of this gene generates several transcript variants encoding different isoforms. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P07288 |
| NCBI GenInfo Identifier: | 130989 |
| NCBI Gene ID: | 354 |
| NCBI Accession: | P07288.2 |
| UniProt Secondary Accession: | P07288,Q15096, Q16272, Q86TG8, Q8IXI4, C9JXH3, G3V0H4 G3XAE3, |
| UniProt Related Accession: | P07288 |
| Molecular Weight: | 28,741 Da |
| NCBI Full Name: | Prostate-specific antigen |
| NCBI Synonym Full Names: | kallikrein-related peptidase 3 |
| NCBI Official Symbol: | KLK3 |
| NCBI Official Synonym Symbols: | APS; PSA; hK3; KLK2A1 |
| NCBI Protein Information: | prostate-specific antigen; seminin; P-30 antigen; kallikrein-3; semenogelase; gamma-seminoprotein; prostate specific antigen |
| UniProt Protein Name: | Prostate-specific antigen |
| UniProt Synonym Protein Names: | Gamma-seminoprotein; Seminin; Kallikrein-3; P-30 antigen; Semenogelase |
| Protein Family: | Prostate-specific antigen |
| UniProt Gene Name: | KLK3 |
| UniProt Entry Name: | KLK3_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-Prostate-specific Antigen 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.)
Related Products
