The Method of Preparing Western Sample —Protein Extraction

  Sample preparation is the first step of Western experiments, which is also a key to the experiment. The obtained protein samples must be homogeneous, soluble, and dissociated into individual peptide subunits, and minimize the aggregation among each other so that they eventually rely only on their own molecular weight size for separation. Common sources of Western samples are recombinant proteins, cells and tissues, etc. The basic steps can be divided into three steps: first, obtain the material and extract the target protein; second, prepare the protein quantification; third, prepare the sample for electrophoresis. First of all, we will introduce the first step: protein extraction.

General principles and considerations for protein extraction.

1. Extract as completely as possible or reduce the complexity of the sample by concentrating only on the target protein.

2. Keep the protein in a soluble state (by the choice of pH salt concentration of the lysate, surfactants, reducing agents, etc.).

3. Prevent the protein degradation, aggregation, precipitation, modification, etc. (low temperature operation, addition of appropriate protease and phosphatase inhibitors) during the extraction process.

4: Remove the interfering molecules such as nucleic acids, polysaccharides, lipids, etc., as much as possible (by adding nucleases or adopting different extraction strategies).

5: Divide and store the samples at -80℃ for a long time to avoid repeated freezing and thawing.

Cell lysis

1. Rinse the cultured cells twice with pre-chilled PBS and supplement the lysate with protease and phosphatase inhibitors.

2. Aspirate the PBS and add the pre-cooled lysis solution (1 ml per 107 cells/100 mm dish/150 cm2 flask).

3. Scrape the adnexal cells with a cell scraper and gently transfer the cells and lysate to a pre-cooled microcentrifuge tube.

4. Shake at 4°C for 30mins.

5. Centrifugate at 4°C and 12,000 rpm for 20mins.

6. Gently aspirate the supernatant and transfer to a freshly pre-cooled microcentrifuge tube on ice for the protein sample and discard the precipitate.

Tissue lysis

1. Separate the target tissue with a sterilized, pre-cooled tool, placed on ice as much as possible to prevent protease hydrolysis.

2. Place the tissue in round-bottom microcentrifuge tubes or EP tubes and adding liquid nitrogen to freeze the tissue for homogenization and grinding on ice, which can be stored at -80°C for a long time.

3. Add approximately 300 ul of pre-cooled lysate per 5 mg, homogenize in an ice bath and place at 4°C for 2 hours with a proper ratio of lysate volume to tissue sample volume (final protein concentration of at least 0.1 mg/ml, ideal protein concentration should be 1-5 mg/ml).

4. Centrifuge at 12,000 rpm for 20mins at 4°C. Gently aspirate the supernatant and transfer to a freshly pre-cooled microcentrifuge tube on ice (i.e., protein sample) and discard the precipitate.