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Dry information sharing | Extraction of cell protein

Publish Time: 2024-07-15     Origin: Site

Protein extraction is the first step in doing a WB experiment. In order to ensure the quality and stability of the protein, it is necessary to maintain low temperature throughout the extraction process and pre-cool the centrifuge and the centrifuge tube containing the protein in advance.


1. Cell plating

Use a 6cm small dish for cell plating, mark the cover and bottom, and shake the cells evenly using the '∞' method. Samples were collected when the cell density reached 90%.



It is recommended to use Aijin 6cm cell culture dish. After TC treatment, the cell adhesion effect is good.

2. Collect samples


It's almost enough if you look like this

Discard the old culture medium and wash twice with PBS (the cells are still viable at this time and there is no need to pre-cool the PBS). Use a pipette tip to absorb the remaining liquid and place the culture dish on ice to pre-cool. If the cells grow at different rates, the samples can be placed at -80°C first and then extracted after all samples are collected.


3. lysis


Lyse on ice for 15 minutes
and add 120 μL of lysis buffer to a 6cm small dish (you can use the lysis buffer without adding PMSF directly. If you use RIPA lysis buffer, you need to add PMSF at a ratio of 100:1 and prepare it now). Gently shake the small dish to evenly distribute the lysis solution, and lyse on ice for 15 minutes. During lysis, label and place 1.5 mL centrifuge tubes on ice to pre-cool, and pre-cool the centrifuge to 4°C. Prepare cell scrapers (it is recommended to choose a cell scraper with a wider scraper head for higher scraping efficiency).

Label the 1.5ml centrifuge tube and place on ice to pre-cool


4. Scrape protein


Use a cell scraper to scrape off the protein to the edge, use a gun to transfer it to a pre-chilled 1.5mL centrifuge tube. Place an ice brick under the petri dish to keep the temperature low while scraping the egg whites.

5. Ultrasound


Generally, 3-5 times of ultrasound are performed. Heat will be generated during the ultrasound process. Therefore, the centrifuge tube must be placed on ice or placed in an ice box to cool down immediately after the ultrasound is completed. If there is no ultrasonic machine, you can use a vortex mixer to mix for 60 seconds.


6. Centrifuge and remove the supernatant


Centrifuge at 12,000 g for 6 minutes at 4°C, and transfer the supernatant to a new, pre-cooled centrifuge tube.


7. Protein quantification and loading


Use the BCA method to determine the protein concentration and calculate the loading volume. The general loading volume is 30 μg.


8. Cook samples


Add 5×Loading Buffer in a ratio of 4:1 and vortex to mix thoroughly. Cook in a metal bath at 100°C for 5 minutes and immediately cool on ice after cooking.


9. Save


If not used immediately, samples should be promptly stored at -20°C.



Protein extraction is the key to ensuring the success of the experiment. Strictly maintaining low temperature, selecting appropriate experimental equipment, and standardizing the operation are all very important. It is recommended to use Aijin 6cm cell culture dish. The product has undergone strict quality control, which can effectively improve the quality and stability of the protein and provide a reliable basis for subsequent experiments. Choose compliant products to make experiments more efficient and smoother!


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