Experimental Steps of Western Blotting Test

Western blot is a protein detection and analysis technique widely used in the fields of biology and medicine. It can be used to detect the expression level of specific proteins in tissue homogenates or cell extracts.

Western blotting technology steps are mainly divided into two parts, namely SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.

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The main principle of the former is that proteins of different molecular weights move at different rates in the gel to separate them.

The latter uses an electric field to transfer the protein from the gel to the membrane of a special material, and then uses the antigen-antibody reaction: the first antibody specifically binds to the target protein, and then the second antibody of the same species as the primary antibody interacts with the target protein. The primary antibody binds to realize the amplification of the target protein signal, and the secondary antibody is attached with an enzyme or a fluorescent group, and the detection and analysis of the target protein can be realized through a luminescent substrate or direct light emission.

“Basic Principles of Western Blot”

Experimental steps:

1. Extraction and preparation of total cell protein

①　Discard the culture medium, rinse gently with pre-chilled 1XPBS twice

②　Add 1ml of pre-cooled 1XPBS, scrape off the cells with a cell scraper and transfer to a 1.5ml EP tube

③　4℃, 5000rpm, centrifuge for 5min

④　Discard the supernatant, add 4 times the precipitation volume of the lysate (containing protease inhibitor), and fully suspend the pellet.

⑤　4℃ rotary shaker pyrolysis for 30min

⑥　4℃, 14000rpm, centrifugation for 10min, carefully transfer the supernatant to a new EP tube

⑦　BCA method for protein quantification, add Loading Buffer to mix according to the concentration, and bathe at 100℃ for 10min to fully denature the protein.

2. SDS polyacrylamide gel electrophoresis (SDS-PAGE)

1) Preparation of gel:

According to the molecular weight range of the protein, configure the appropriate concentration of SDS-PAGE gel. Each gel needs about 7ml of separation gel and 3ml of concentrated gel. The thin plate and the thick plate are placed on the glue making rack, and the separating glue is added first, and the glue is sealed with isopropanol for 30 minutes to make the glue fully solidify. Pour out the isopropanol and fully blot it dry. Insert the comb immediately after adding the concentrated glue. After about 30 minutes, the comb can be unplugged and used after the glue has solidified.

2) Electrophoresis:

Install the gel plate correctly in the electrophoresis tank, slowly add 1X electrophoresis buffer, slowly add samples to the wells, add the color pre-stained protein Marker on one side, and add a certain volume of protein samples to each well in turn. After the sample is added, connect the power supply, constant voltage 60V, electrophoresis for about 40 minutes (marker starts to separate) and then constant voltage 100V, the time is subject to the required protein separation.

3. Gel electrotransfer of protein

Place the prepared PVDF membrane in a methanol solution for several seconds to activate, and then place the activated PVDF membrane and filter paper in the transfer buffer to equilibrate for a short period of time.

Take out the glass plate from the electrophoresis tank, remove the gel after electrophoresis on the rocker, remove the concentrated gel and cut off the excess separating gel. Follow the sequence of “filter paper-gel-PVDF membrane-filter paper” to make the gel layer close to the negative electrode and the PVDF membrane layer close to the positive electrode to avoid air bubbles between the interlayers.

Then put the entire device into the transfer tank, add the transfer liquid, cool the outside of the transfer tank in an ice water bath, and transfer for 80 minutes at a constant pressure of 100V. After the transfer electrophoresis, the PVDF membrane is taken out.

4. Protein immune response

① 　 Sealing: Immerse the transferred PVDF membrane in 5% skimmed milk powder sealing solution and seal it, shaker for 50-60 revolutions, 2 hours (room temperature).

②    Incubate with primary antibody: select the appropriate concentration to dilute the antibody according to the antibody specification, add the diluted antibody to the antibody incubation box, and put the PVDF membrane into it, and incubate at 4°C overnight with a shaker at 50-60 revolutions. Take it out the next day and wash the membrane with TBST (3 times/15 min, 60 revolutions).

③　Secondary antibody incubation: Dilute the secondary antibody to an appropriate concentration, put the PVDF membrane in the diluted secondary antibody working solution, incubate at room temperature for 1 hour, and shake the bed for 80 revolutions. After the incubation, take out the PVDF membrane and wash the membrane with TBST (3 times/15 min, 80 revolutions)

④　Development: Take appropriate amount of chemiluminescence reagents A and B, mix them in equal volume, add them evenly on the PVDF membrane, incubate for tens of seconds, use a gel imaging system to expose and acquire images. Measure the gray value of the band by the gel imaging system, and calculate the relative expression of the experimental protein.

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