Cytosol Nucleus And Membrane Protein Extraction Kit

This kit offers a straightforward and hassle-free approach to extract various types of proteins and cells from fresh tissues or cultured cells. The protein extraction process, which includes the separation of nuclear proteins, cytoplasmic proteins, membrane proteins and cytoskeleton proteins of cultured cells, can be accomplished within a span of approximately 90 minutes with this kit's framework protein method. This kit is versatile and enables the usage of extracted proteins in various downstream applications like Western, EMSA, footprinting, reporter gene detection and enzyme activity determination. The product is inclusive of cytoplasmic protein extract (60mL), nucleoprotein extract (10mL), membrane protein extract (6mL), structural protein extract (6mL), and phosphatase inhibitor II (50×) (1mL) and FPMSF (100×) (1ml) for optimal results.

experiment protocol:
1. Reagent preparation
1.1 Prepare the solution
Please ensure that the various solutions included in the kit are dissolved at room temperature. Once dissolved, please place all reagents on ice immediately, except for the structural protein extract. Remember to follow this protocol to maintain the integrity of the reagents and ensure accurate experimentation.
Once the structural protein extract has been dissolved, the next step would be to let it sit in a 37°C water bath until it turns transparent. After achieving transparency, it can be taken out of the water bath and left to cool down at room temperature.
To prepare the working solution for protein extraction, there are a few important steps to follow. First, you will need to make sure you have all of the necessary ingredients on hand. These may include buffers, detergents, and protease inhibitors.
Next, you will need to carefully measure out the appropriate amounts of each component and mix them together. It is important to follow the recipe exactly in order to ensure that your working solution is effective.
Once you have prepared your working solution, you can use it to extract proteins from a variety of sources. This may involve subjecting your samples to various physical and chemical treatments in order to break down cell walls and membrane barriers.
Overall, preparing a working solution for protein extraction requires careful attention to detail and a thorough understanding of the underlying biochemical processes involved. With the right approach, however, it is possible to extract high-quality proteins for a variety of research and diagnostic applications.
To prepare the working solution for cytoplasmic protein extraction, first, calculate the amount of cytoplasmic protein extraction solution needed for the experiment based on the number of samples to be processed. Next, add phosphatase inhibitor II and PMSF in a 1:50 ratio, considering the amount of cytoplasmic protein extraction solution. Finally, include the protease inhibitor PMSF in proportion to the other components. This will ensure the appropriate composition of the working solution for effective cytoplasmic protein extraction.
To prepare the working solution of nucleoprotein extract (NPE), it is necessary to estimate the required amount of NPE for the experiment based on the number of processed samples. Additionally, phosphatase inhibitor II and protease inhibitor PMSF should be added in a 1:50 ratio and a 1:100 ratio, respectively. The NPE working solution is crucial for the success of the experiment and should be prepared accurately.
To prepare the working solution for extracting membrane proteins, it is important to estimate the required amount based on the number of samples being processed in the experiment. Additionally, phosphatase inhibitor II should be added in a ratio of 1:50 or 1:100. In order to ensure the integrity of the proteins, it is advisable to include the protease inhibitor PMSF as well.
1.2.4 Preparation of structural protein extraction working solution: before use, estimate the required amount of structural protein extraction solution in the experiment according to the amount of processed samples, and add phosphatase inhibitor II in a ratio of 1:50 and a ratio of 1:100 respectively Protease inhibitor PMSF.
2. For cultured cell samples:
2.1 cytoplasmic protein extraction
2.1.1 Add 2.0ml (2.0×107 cells) of cytoplasmic protein extraction working solution and shake for 20 minutes at 4°C;
2.1.2 Prepare a 1-5ml syringe, use the syringe to blow the solution shaken in step 1 50-90 times, and centrifuge at 15000g for 10 minutes at 4°C. Take the supernatant and store it in the EP tube, which is the cytoplasmic protein;
2.2 Extraction of nucleoprotein
Take the precipitate obtained in step 2.1.2 and add 4.0ml/20million cells ice bath cytoplasmic protein extraction working solution, shake at 4° C for 5 minutes, centrifuge at 15000g at 4° C for 10 minutes, discard the supernatant and precipitate Add 1.0ml/20million cells frozen nucleoprotein extraction working solution, shake for 5 minutes at 4° C, centrifuge at 15000g at 4° C for 10 minutes, the supernatant is nucleoprotein, transfer it to EP tube and save;
2.3 Extraction of membrane proteins
Add 1.0ml/20million cells frozen membrane protein extraction working solution to the precipitate in step 2.2, shake for 5 minutes at 4°C, centrifuge at 15000g at 4°C for 10 minutes, the supernatant is cell membrane protein, transfer it to EP Tube and save.
2.4 Extraction of structural protein
Add 0.5ml/20million cells of a transparent structural protein extraction working solution that has been bathed in water at room temperature or 37°C to the precipitate of step 2.3, shake for 5 minutes at 4°C, centrifuge at 15000g for 10 minutes at 4°C, and save the supernatant. Is a cytoskeleton protein;
2.5 Storage of the protein to be tested
The protein to be tested is stored at -70°C.
3. For tissue samples:
3.1 cytoplasmic protein extraction
3.1.1 Add 2.0ml/g of cytoplasmic protein extraction working solution, grind at 4° C, repeat homogenization twice, until all is dissolved;
3.1.2 At 4°C, shake for 5 minutes and centrifuge at 18000g for 10 minutes. Take the supernatant and store it in the EP tube, which is the cytoplasmic protein, and continue the experiment with the precipitate;
3.2 Extraction of nucleoprotein
Add 4.0ml/g of cytoplasmic protein extraction working solution to the precipitate in step 3.1.2, shake for 5 minutes at 4°C, centrifuge at 18000g for 10 minutes at 4°C, and discard the supernatant; Add 1.0ml/g frozen nucleoprotein extraction working solution, shake at 4° C for 5 minutes, centrifuge at 18000g at 4° C for 10 minutes, the supernatant is nucleoprotein, transfer it to EP tube and save;
3.3 Extraction of membrane proteins
Add 1.0ml/g of frozen membrane protein extraction working solution to the precipitate in step 3.2, shake at 4° C for 5 minutes, centrifuge at 18000g at 4° C for 10 minutes, the supernatant is cell membrane protein, transfer it to EP tube And save
3.4 Extraction of structural proteins
3.4.1 Add 0.5ml/g of transparent structural protein extraction working solution that has been bathed in water at room temperature or 37°C to the precipitate in step 3.3, shake for 5 minutes, centrifuge at 18000g at 4°C for 10 minutes, and save the supernatant;
3.4.2 Add 1.5ml/g frozen cytoplasmic protein extraction working solution to the precipitate in step 3.4.1, shake for 5 minutes at 4° C, centrifuge at 18000g for 10 minutes at 4° C, and save the supernatant;
2.4.3 Mix the supernatant from the first two steps, this is the backbone protein;
3.5 Storage of the protein to be tested
The protein to be tested is stored at -70°C.

Store at -20°C for 12 months

1. In order to obtain the best use effect, try to avoid too many repeated freezing and thawing. It is suggested that it can be used after proper packaging;
2. All utensils and reagents that contact the sample should be pre-cooled. All steps of protein lysis should be performed in an ice bath or 4°C; 3. The reagents in the kit are sufficient to treat 5g tissue, or 1.25×108 cells, and the minimum cells should not be less than 0.1g tissue or 2.5×106.
4. The structural protein extract contains SDS, and it can be dissolved by heating in a precipitation water bath during use.
For your own safety, before using the reagents, please take care, such as wearing lab coats and wearing gloves.