Description
| Product Name: | Human PDE4 (Phosphodiesterase 4, cAMP Specific) ELISA Kit |
| SKU: | AEFI01434 |
| Size: | 96T |
| Reactivity: | Human |
| Range: | 31.25-2000pg/ml |
| Sensitivity: | 18.75pg/ml |
| Detection Method: | Sandwich ELISA, Double Antibody |
| Detection Wavelength: | OD450 |
| Reaction Duration: | 4 hours |
| Synonyms: | PDE4 ELISA Kit, Phosphodiesterase 4, cAMP Specific ELISA Kit |
| Kit Components: |
| |||||||||||||||||||||||||||||||||||||||||||||||||
Other Materials Required (Not provided)
- Microplate reader (wavelength: 450nm)
- 37°C incubator (CO2 incubator for cell culture is not recommenced.)
- Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
- Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
- Sterile tubes and Eppendorf tubes with disposable tips
- Absorbent paper and loading slot
- Deionized or distilled water
This kit is based on Double antibody-Sandwich ELISA detection method and takes 4h assay time. The microplate provided in this kit has been precoated with anti PDE4 antibody. Add standard and properly diluted sample into relevant well respectively. After incubation, wash unbound components. Add biotinylated detection antibody. Then, it binds with PDE4 bound to precoated antibody. Wash unbound components and add HRP-Streptavidin Conjugate (SABC). Wash unbound components again and add TMB substrate solution. Then, TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. Calculate the concentration of PDE4 in the sample by plotting standard curve. The concentration of the target substance is proportional to the OD450 value.
| Recovery: | Add a certain amount of PDE4 into the sample. Calculate the recovery by comparing the measured value with the expected amount of PDE4 in the sample.
| ||||||||||||||||||||||||||||||||||||||||||
| Precision: | Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate. Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.
| ||||||||||||||||||||||||||||||||||||||||||
*Note:The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Protocol |
| 1. | Add 100ul standard or sample into each well, seal the plate and statically incubate for 90 minutes at 37°C. |
| 2. | Washing: Wash the plate twice without immersing. |
| 3. | Add 100ul biotin-antibody working solution, seal the plate and statically incubate for 60 minutes at 37°C. |
| 4. | Washing: Wash the plate three times and immerse for 1min each time. |
| 5. | Add 100ul HRP-Streptavidin Conjugate (SABC) working solution, seal the plate and statically incubate for 30 minutes at 37°C. |
| 6. | Washing: Wash the plate five times and immerse for 1min each time. |
| 7. | Add 90ul TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.) |
| 8. | Add 50ul stop solution. Read at 450nm immediately and calculate. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Tissue Sample | Generally tissue samples are required to be made into homogenization. Protocol is as below: 1.1. Place the target tissue on the ice. Remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Then weigh for usage. 1.2. Use lysate to grind tissue homogenates on the ice. The adding volume of lysate depends on the weight of the tissue. Usually, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS (e.g. 1mM PMSF). 1.3. Do further process using ultrasonic disruption or freeze-thaw cycles (Ice bath for cooling is required during ultrasonic disruption; Freeze-thaw cycles can be repeated twice.) to get the homogenates. 1.4. Homogenates are then centrifuged for 5 minutes at 5000×g. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. 1.5. Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for Elisa assay should be within 1-3mg/ml. Some tissue samples such as liver, kidney, pancreas which containing a higher endogenous peroxidase concentration may react with TMB substrate causing false positivity. In that case, try to use 1% H2O2 for 15min inactivation and perform the assay again. Notes:PBS buffer or the mild RIPA lysis can be used as lysates While using RIPA lysis, make the PH=7.3. Avoid using any reagents containing NP-40 lysis buffer, Triton X-100 surfactant, or DTT due to their severe inhibition for kits’ working. We recommend using 50mM Tris+0.9%NaCL+0.1%SDS, PH7.3. You can prepare by yourself or contact us for purchasing. |
| Cell Culture Supernatant | Collect the supernatant: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes, then collect clarified cell culture supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay.. |
| Cell Lysate | 3.1.Suspension Cell Lysate: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes and collect cells. Then add pre- cooling PBS into collected cell and mix gently. Recollect cell by repeating centrifugation. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Lyse the cell on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 3.2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS to wash three times. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape the adherent cell with cell scraper. Lyse the cell suspension added in the centrifuge tube on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 3.3. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Mucilaginous product is DNA which can be disrupted by ultrasonic cell disruptor on ice. (3~5mm probe, 150-300W, 3~5 s/time, 30s intervals for 1~2s working). 3.4. At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube to detect immediately. Or you can aliquot the supernatant and store it at - 80°C for future’s assay. Notes:Read notes in tissue sample. |
| Other Biological Sample | Centrifuge samples for 15 minutes at 1000×g at 2-8℃. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay.. |
| Sample Type: | Cell or tissue lysate, Other liquid samples |
| Storage: | 2-8°C(Sealed), Don't cryopreserve. |
| Specificity: | Specifically binds with PDE4 , no obvious cross reaction with other analogues. |
