Cytoplasmic and Membrane Protein Separation and Extraction Kit servicebio G2280-50T
$50.00
In stock
SKU
G2280-50T
Product Information
| Product Name | Cat. No. |
| Spec. | Cytoplasmic and Membrane Protein Separation and Extraction Kit |
| G2280 | 50T |
Product Introduction
This kit provides a simple and convenient method for separating and extracting plasma and membrane proteins (applicable to the separation and extraction of cell plasma and membrane proteins in cells and tissues), which can be completed within 60 minutes. Different component proteins can be extracted from the same sample separately and used to study the localization of different components in cells. The separated protein can be used for subsequent experiments such as SDS-PAGE, WB, and activity detection.
This product uses a specific plasma protein extraction reagent to lyse cells and centrifuge to obtain cytoplasmic proteins. The obtained precipitate was dissolved using membrane protein extraction reagent to obtain all membrane component proteins (including membrane proteins of organelles such as cell membrane, endoplasmic reticulum, mitochondria, etc.).
Storage and Shipping Conditions
Shipping with dry ice；Store at -20℃ and valid for 12 months.
Component
| Component number | Component |
| G2280 | G2280-1 |
| Cytoplasmic Protein Extraction buffer | 15 mL |
| G2280-2 | Membrane Protein Extraction reagent |
| 10 mL | Manual |
| 1 pc |
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 and 2% by volume to the Cytoplasmic Protein Extraction Buffer.add prior to use to avoid failure.
1.3. Cell lysis: Add Cytoplasmic Protein Extraction working fluid according to the total amount of cells. It is recommended to add 300 μL of plasma protein extraction working solution for 5×10^6 total cells (different cell sizes vary, it is recommended to add plasma protein extraction working solution according to the amount of cell precipitation, and add 100 μL of plasma protein extraction working solution for every 10 μL of cell precipitation. For example, the Hela precipitation amount of 10^6 is about 10 μL, and add 100 μL of plasma protein extraction working solution). Resuspend cells and lyse at 4 ℃ for 10 minutes. Vortex 2-3 times during the cracking process.
1.4. Centrifuge at 4 ℃ 12000 g， for 5 minutes, and collect the supernatant to obtain cytoplasmic protein.
1.5. Membrane protein extraction: Use pre cooled PBS to vortex resuspend and wash the precipitate of 2.4. After washing, centrifuge at 12000 g at 4 ℃ for 5 minutes, discard the supernatant, and try to absorb the liquid as much as possible to prevent protein contamination. Add 200 μL of Membrane Protein Extraction working fluid to each tube, resuspend the precipitate, and incubate at 4 ℃ for 10 minutes. Vortex 2-3 times during incubation process.
1.6. Centrifuge at 12000 g and 4 ℃ for 5 minutes, and collect the supernatant to obtain the membrane 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 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 300 μL of Cytoplasmic Protein Extraction working fluid per 20 mg of tissue and homogenise thoroughly using a low-temperature tissue grinder. Set the parameters for the SWE-FP grinder to 60 Hz, 30 seconds, twice.
2.4. Centrifuge at 4 ℃ 12000 g， for 5 minutes, and collect the supernatant to obtain cytoplasmic protein.
2.5. Membrane protein extraction: Use pre cooled PBS to vortex resuspend and wash the precipitate of 2.4. After washing, centrifuge at 12000 g at 4 ℃ for 5 minutes, discard the supernatant and absorb the liquid as much as possible to prevent protein contamination. Add 200 μL of Membrane Protein Extraction working fluid to each tube, resuspend the tissue precipitate, and incubate at 4 ℃ for 10 minutes. Vortex 2-3 times during incubation process.
2.6. Centrifuge at 12000 g and 4 ℃ for 5 minutes, and collect the supernatant to obtain the membrane 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 entire experiment needs to be conducted in a low-temperature environment.
3. Membrane proteins tend to aggregate when boiled at high temperatures. For transmembrane proteins, the reference denaturation temperature is 50-70 ℃ for 15 minutes or 37 ℃ for 30 minutes.
4. Phosphatase inhibitors can be added to protein extraction reagents according to experimental requirements
5. The precipitated sample needs to be resuspended thoroughly to achieve better extraction efficiency
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.
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