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Storage:Store PMSF at -20℃, the rest at 4℃, valid for 1 year.
Product Introduction:
This nucleoprotein extraction kit provides a simple and convenient method for extracting nucleoprotein from cultured cells or fresh tissues. The whole process takes only about 60 minutes to separate the nuclear and plasma proteins from the cell. The resulting proteins can be used in experiments such as Western blot. This kit uses plasma protein extraction reagent to make the cell expand and rupture, release the cytoplasmic protein, and then obtain the nucleus by centrifugation. Then nuclear protein was extracted by nuclear protein extraction reagent. This kit can extract 50/100 samples (about 2×106 Hela cells or 40mg tissue per sample).
Kit Components:
试剂盒组成 | 50T | 100T |
浆蛋白抽提试剂 | 25ml | 50ml |
核蛋白抽提试剂 | 5ml | 10ml |
PMSF（100mM） | 300μl | 1ml |
How to operate: All the steps of cracking protein should be carried out on ice or at 4 ℃.
1. Appropriate plasma protein extraction reagent and nuclear protein extraction reagent were added to PMSF according to the dosage, so that the final concentration of PMSF was 1mM. PMSF should be used on the spot. If the target protein is rich in cysteine, DTT can be added to the two protein extracts to the final concentration of 0.5mM.
2. For adherent cells, the culture medium is removed, the cells are washed with PBS, the cells are collected with a cell scraper, or the cells are blown down after digestion with EDTA (it is better not to nitrate with pancreatic enzyme, so that pancreatic enzyme does not digest the target protein). Centrifuge at 500 g for 2 ~ 3 minutes to collect cells, suck up the supernatant and leave the precipitation for later use.
3. For suspended cells: Wash with PBS, centrifuge at 500 g for 2 ~ 3 minutes to collect the cells, suck up the supernatant, and leave the cell precipitation for later use.
4. 200μl plasma protein extraction reagent was added for every 20μl cell precipitate (the volume of 2×106 cell precipitate was about 20μl or 40mg).
5. By blowing with a pipette or swirling at a high speed for 15 seconds (which can be extended appropriately), the cell precipitates must be completely dispersed into a single-cell suspension. Incomplete cell precipitation and dispersion will result in decreased protein production.
6. Ice bath for 10 minutes.
Seven. Maximum speed of intense vortex for 10 seconds, 4℃ 12000 ~ 16000g centrifugation for 10 minutes.
8. The supernatant is the extracted cytoplasmic protein, which is immediately absorbed into the pre-cooled sample tube for use in subsequent PAGE, Western and other experiments, but the protein concentration is low, and can be concentrated accordingly according to needs.
9. Precipitation is the nucleus, to completely absorb the residual supernatant (to avoid cytoplasmic protein contamination), add 50-100μl nuclear protein extraction reagent.
10. Use a pipette to blow or vortex at high speed for 15 seconds (can be extended appropriately) until the precipitate is completely dispersed, and ice bath for 10 minutes.
11. Maximum speed of intense vortex for 10 seconds, 4℃12000 ~ 16000g centrifugation for 10 minutes. 12. Immediately absorb the supernatant into the pre-cooled sample tube, and this is the extracted nuclear protein. Follow-up experiments can be performed or frozen at -70℃. For tissue samples: After weighing the tissue, cut it into very fine pieces as much as possible, add 200μl-500μl PBS per 50mg tissue, homogenize it on ice to make cell suspension, centrifuge it at 500 g for 2 to 3 minutes to collect cells, suck up the supernatant, and collect precipitation. Add 200μl of plasma protein extraction reagent and follow the above step 5.
Note:
1. The protein sample obtained by cracking can not be determined by Bradford method due to the interference of relatively high concentration of detergent. The BCA protein quantitative kit (item No. PC0020) produced by our company can be used to determine the protein concentration.
2. For tissue samples, this kit is suitable for fresh tissue, and has poor extraction effect on frozen tissue.
3. For your safety and health, please wear a lab coat and disposable gloves.
Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.
