| Product Description | This product can rapidly, gently, and efficiently lyse mammalian cells, effectively extracting cytoplasmic and nuclear proteins. This reagent uses a mild formula to ensure that the extracted protein maintains biological activity and can be applied to various protein analysis experiments, such as reporter gene and enzyme activity determination, immune detection, protein purification, etc. The extracted protein can be quantitatively analyzed using the BCA method. The reagent kit contains a mixture of protease inhibitors, which can effectively prevent protein degradation during the protein extraction process. | M665813 | Component | 100 T | Storage | | M665813A | Mammalian Protein Extraction Reagent | 100 mL | RT | | M665813B | Protease Inhibitor Cocktail (100×) | 1 mL | -20℃. Avoid freeze/thaw cycle. |
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precautions
1. This product can effectively lyse adherent cells cultured on cell culture plates (without scraping) and suspended cells collected by centrifugation, with higher extraction efficiency than repeated freeze-thaw or ultrasound methods. But for the extraction of tissue proteins, it is recommended to use the tissue protein extraction kit (CW0891).
The optimal dosage for protein extraction from adherent cells is listed in Table 1. Collecting cells first can reduce the amount of reagents used to obtain higher protein concentrations.
3. The amount of extraction reagents used can also be estimated based on the number of cells. If 2 × 106 Hela cells weigh about 20 mg, 200 need to be added μ Extract reagents.
4. The protein extracted from this product can be quantitatively analyzed using the BCA method. Operation steps
● Protein extraction from adherent cells
1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.
2. Carefully pour out the culture medium of adherent cells and rinse the cells with PBS.
3. Add an appropriate amount of Mammalian Protein Extraction Reagent (add Protein Inhibitor Cocktail in a 1:99 ratio 2-3 minutes before protein extraction), blow adherent cells on ice with a gun tip, transfer the lysate to a centrifuge tube, incubate on ice for 20 minutes, and allow the cells to fully lyse (please refer to Appendix 1 for the amount of reagent used, and the time for placing on ice should be adjusted according to different cell types).
4. Centrifuge at 14000 × g for 5-10 minutes.
5. Transfer the supernatant to a new tube for further analysis.
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Suspension cell protein extraction
1. Please remove the required Mammalian Protein Extraction Agent for pre cooling before protein extraction.
2. Suspend 2500 × g of cells, centrifuge for 10 minutes, and discard the supernatant. Rinse cells with PBS. 2500 × g, centrifuge for 10 minutes, discard the supernatant.
3. Add an appropriate amount of Mammalian Protein Extraction Agent, and 2-3 minutes before protein extraction, add Protein Inhibitor Cocktail in a ratio of 1:99, which is 1 x working solution.
4. Add at least 1 ml of 1x working solution to every 100 mg of cells. If the extracted sample size is large, a small amount of 1x working solution can be used to resuspend the cells first, and then the remaining working solution can be added.
5. After blowing evenly, place it on ice for 20 minutes to allow the cells to fully lyse (the time for placing it on ice should be adjusted according to different cell types).
6. Centrifuge at 14000 × g for 15 minutes.
7. Transfer the supernatant to a new tube for further analysis. Table 1. Recommended usage of extraction reagents
| Cell culture plate type or dish type |
Extraction reagent usage |
| 100 mm |
500-1,000 µl |
| 60 mm |
250-500 µl |
| 6-well culture plate |
200-400 µl /well |
| 24-well culture plate |
100-200 µl /well |
| 96-well culture plate |
50-100 µl /well |
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Table 2. Common Problems and Solutions
| Problem |
Possible reasons |
Resolvent |
| Low extraction rate |
Low protein expression level |
Optimize transfection system |
| Low extraction rate |
Insufficient reagent usage |
Increase the usage of extraction reagents |
| Low extraction rate |
Reagent unable to dissolve cell membrane |
Increase cracking time or increase shaking amplitude |
| Unable to obtain membrane protein |
This product is more suitable for extracting nuclear plasma protein |
Using eukaryotic cell membrane protein extraction kit | |
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