Cytoplasmic, Membrane and Nuclear proteins Separation and Extraction Kit servicebio G3742-50T
$50.00
In stock
SKU
G3742-50T
Product Information
| Product Name | Cat.No. |
| Spec. | Cytoplasmic, Membrane and Nuclear proteins Separation and Extraction Kit |
| G3742 | 50T |
Product Itroduction
This product is suitable for the separation and extraction of Cytoplasmic, Membrane and Nuclear proteins, and can be used to study the localisation of different proteins in cells. The separated proteins can be used for SDS-PAGE, WB, EMSA and other experiments.
This kit provides a simple, convenient and fast method for separating and extracting of Cytoplasmic, Membrane and Nuclear proteins. It is able to finish this process in 90min.
This product uses specific Cytoplasmic extraction buffer to lyse cells, centrifugation to obtain supernanent as cytoplasmic proteins.Then using membrane protein extraction buffer to obtain all membrane component proteins in precipitate (containing membrane proteins of cell membranes, endoplasmic reticulum, mitochondria and other organelles).Finally, cellular nuclear proteins were fully extracted using Nuclear Protein Extraction Reagents A and B.
Storage and Shipping Conditions
Shipping with dry ice；Store at -20℃ and valid for 12 months.
Component
| Component number | Component |
| G3742 | G3742-1 |
| Cytoplasmic Protein Extraction buffer | 15ml |
| G3742-2 | Membrane Protein Extraction buffer |
| 10ml | G3742-3 |
| Nuclear Protein Extraction buffer A | 5ml |
| G3742-4 | Nuclear Protein Extraction buffer B |
| 250μl | Manual |
| 1pc |
Assay Protocol
1. Cell ：
1.1. Rewarming this product at room temperature and place on ice before use;
1.2. Prepare the working fluid:
1.2.1. Cytoplasmic Protein Extraction working fluid：Add Cocktail, PMSF to the Cytoplasmic Protein ExtractionBuffer.add prior to use to avoid failure.
1.2.2. Membrane Protein Extraction working fluid:Add Cocktail, PMSF to the Cytoplasmic Protein ExtractionBuffer.add prior to use to avoid failure.
1.2.3. Nuclear Protein Extraction working fluid：Add Cocktail, PMSF and 2% by volume to the Cytoplasmic Protein Extraction Buffer.add prior to use to avoid failure.
1.3. Cell lysis：According to the volume of cell precipitation, add Cytoplasmic Protein Extraction working fluid. resuspend the cells at 4°C, and lysed for 10 min. vortex 2-3 times during the lysis process. The volume of lysate to be used is determined according to the volume of cell precipitation, and it is recommended to add 100 μl of working fluid to 10 μl of cell precipitation. For example, the volume of 10^6 total Hela precipitation is about 10μl, Add 100μl of cytoplasmic protein extraction working fluid.
1.4. The supernatant was collected by centrifugation at 4°C, 2000g, 5min.removed supernatant to reduce contamination of cytoplasmic proteins in nuclear protein extraction.
1.5. Recentrifugation the supernanent in step 1.4 at 4°C, 2000g, 5min,collecting the supernanent as cytoplasmic protein.
1.6. Membrane Protein Extraction:Using 1ml of pre-cooled PBS resuspened and wash the precipitate in step 1.4. Then centrifuge at 2000g for 5min at 4℃, discard the supernatant and try to aspirate the liquid to prevent protein contamination. Add 200μl of cell membrane protein extraction working fluid to each tube, resuspend the precipitate, and incubate at 4℃ for 10min. vortex 2-3 times during the incubation.
1.7. 2000g, centrifuge at 4℃ for 5min, collect the supernatant for membrane protein extraction and precipitate for nuclear protein extraction.
1.8. Recentrifugation the supernanent in step 1.7 at 4°C, 2000g, 5min,collecting the supernanent as Membrane protein.
1.9. Nuclear protein extraction:Using 1ml of pre-cooled PBS resuspened and wash the precipitate in step 1.7. Then centrifuge at 2000g for 5min at 4℃, discard the supernatant and try to aspirate the liquid to prevent protein contamination.Add 50-100 μl of Cytosolic Protein Extraction working fluid to the precipitate Vortex and resuspend, and extract for 30min at 4°C, vortex for 10-15s every 3min.
1.10. The supernatant was collected by centrifugation at 4°C, 2000g, 5min. as nuclear protein.
2. Fresh Tissue or Frozen Tissues：
2.1. Rewarming this product at room temperature and place on ice before use;
2.2. Prepare the working fluid:
2.2.1. Cytoplasmic Protein Extraction working fluid：Add Cocktail, PMSF to the Cytoplasmic Protein ExtractionBuffer.add prior to use to avoid failure.
2.2.2. Membrane Protein Extraction working fluid:Add Cocktail, PMSF to the Cytoplasmic Protein ExtractionBuffer.add prior to use to avoid failure.
2.2.3. Nuclear Protein Extraction working fluid：Add Cocktail, PMSF and 2% by volume to the Cytoplasmic Protein Extraction Buffer.add prior to use to avoid failure.
2.3. slice the tissue into as fine pieces as possible and wash 1-2 times using pre-cooled PBS. Add 1 mL of Cytoplasmic Protein Extraction working fluid per 50 mg of tissue and homogenise well using a pre-cooled glass homogeniser or a Tissue Homogenizer Low Temperature(SWE-FP).Homogenise well using Homogenizer.Grind 30-60 times with the glass homogeniser and set the parameters of the Tissue Homogenizer Low Temperature to 60hz, 30s, 2 times.Grinding conditions can be adjusted according to tissue type.
2.4. The supernatant was collected by centrifugation at 4°C, 2000g, 5min.removed supernatant to reduce contamination of cytoplasmic proteins in nuclear protein extraction.
2.5. Recentrifugation the supernanent in step 1.4 at 4°C, 2000g, 5min,collecting the supernanent as cytoplasmic protein.
2.6. Membrane Protein Extraction:Using 1ml of pre-cooled PBS resuspened and wash the precipitate in step 1.4. Then centrifuge at 2000g for 5min at 4℃, discard the supernatant and try to aspirate the liquid to prevent protein contamination. Add 200μl of cell membrane protein extraction working fluid to each tube, resuspend the precipitate, and incubate at 4℃ for 10min. vortex 2-3 times during the incubation.
2.7. 2000g, centrifuge at 4℃ for 5min, collect the supernatant for membrane protein extraction and precipitate for nuclear protein extraction.
2.8. Recentrifugation the supernanent in step 2.7 at 4°C, 2000g, 5min,collecting the supernanent as Membrane protein.
2.9. Nuclear protein extraction:Using 1ml of pre-cooled PBS resuspened and wash the precipitate in step 2.7. Then centrifuge at 2000g for 5min at 4℃, discard the supernatant and try to aspirate the liquid to prevent protein contamination.Add 50-100 μl of Cytosolic Protein Extraction working fluid to the precipitate Vortex and resuspend, and extract for 30min at 4°C, vortex for 10-15s every 3min.
2.10. The supernatant was collected by centrifugation at 4°C, 2000g, 5min. as nuclear protein.
Note
1. The reagents are stored at -20℃ and need to be pre-cooled in an ice bath immediately after thawing at room temperature before use.
2. The whole experiment should be carried out in a low-temperature
3. Membrane proteins are prone to aggregation and formation of dimers or multimers when boiled at high temperatures. For transmembrane proteins, the reference denaturation temperature is 50-70℃ for 15min or 37℃ for 30min.
4. Phosphorylated protease inhibitors can be added to the protein extraction reagent according to experimental requirements.
5. Try to resuspend the precipitate when extracting membrane protein and nuclear proteins in order to get a better extraction effect.
6. Add PMSF before use to avoid failure.
7. This product is suitable for fresh or frozen tissues, but the separation and extraction efficiency of frozen tissues may decrease.
8. For your safety and health, please wear lab coat and disposable gloves
For Research Use Only!
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