Description
PPIF Colorimetric Cell-Based ELISA
The PPIF (Peptidylprolyl Isomerase F) Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate detection of PPIF levels in cell lysates and cell culture supernatants. This kit features a simple and efficient colorimetric assay, allowing for easy quantification of PPIF levels in various cell types.PPIF, also known as cyclophilin D, plays a critical role in mitochondrial function and cell death pathways. Dysregulation of PPIF has been implicated in various diseases, including neurodegenerative disorders, cardiovascular diseases, and cancer.
By accurately measuring PPIF levels, researchers can gain valuable insights into the mechanisms underlying these diseases and potentially identify new therapeutic targets.The PPIF Colorimetric Cell-Based ELISA Kit offers high sensitivity and specificity, ensuring reliable and reproducible results. With its user-friendly protocol and quick assay time, this kit is an essential tool for researchers studying mitochondrial function, cell death pathways, and disease mechanisms.
| Product Name: | PPIF Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00998 |
| ELISA Type: | Cell-Based |
| Target: | PPIF |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The PPIF Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PPIF protein expression profile in cells. The kit can be used for measuring the relative amounts of PPIF in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on PPIF.
Qualitative determination of PPIF concentration is achieved by an indirect ELISA format. In essence, PPIF is captured by PPIF-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: 10105, UniProt ID: P30405, OMIM: 604486, Unigene: Hs.381072 |
| Gene Symbol: | PPIF |
| Sub Type: | None |
| UniProt Protein Function: | PPIF: PPIases accelerate the folding of proteins. It catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides. Belongs to the cyclophilin-type PPIase family. |
| UniProt Protein Details: | Protein type:EC 5.2.1.8; Isomerase; Mitochondrial Chromosomal Location of Human Ortholog: 10q22.3 Cellular Component: membrane; mitochondrial matrix; mitochondrial proton-transporting ATP synthase complex Molecular Function:peptidyl-prolyl cis-trans isomerase activity; protein binding Biological Process: negative regulation of apoptosis; negative regulation of ATPase activity; regulation of mitochondrial membrane permeability |
| NCBI Summary: | The protein encoded by this gene is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. PPIases catalyze the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. This protein is part of the mitochondrial permeability transition pore in the inner mitochondrial membrane. Activation of this pore is thought to be involved in the induction of apoptotic and necrotic cell death. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P30405 |
| NCBI GenInfo Identifier: | 231968 |
| NCBI Gene ID: | 10105 |
| NCBI Accession: | P30405.1 |
| UniProt Secondary Accession: | P30405,Q2YDB7, Q5W131, |
| UniProt Related Accession: | P30405 |
| Molecular Weight: | 16,541 Da |
| NCBI Full Name: | Peptidyl-prolyl cis-trans isomerase F, mitochondrial |
| NCBI Synonym Full Names: | peptidylprolyl isomerase F |
| NCBI Official Symbol: | PPIF |
| NCBI Official Synonym Symbols: | CYP3; CypD; CyP-M; Cyp-D |
| NCBI Protein Information: | peptidyl-prolyl cis-trans isomerase F, mitochondrial |
| UniProt Protein Name: | Peptidyl-prolyl cis-trans isomerase F, mitochondrial |
| UniProt Synonym Protein Names: | Cyclophilin D; CyP-D; CypD; Cyclophilin F; Mitochondrial cyclophilin; CyP-M; Rotamase F |
| Protein Family: | Peptidyl-prolyl cis-trans isomerase |
| UniProt Gene Name: | PPIF |
| UniProt Entry Name: | PPIF_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-PPIF 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.)
