Western Blot sample preparation is the first step in this experiment. And it is a key step that requires getting all the protein possible. Therefore, we should pay attention to the following problems when doing wtstern blot experiments:

1: The maximum solubility and reproducibility of the protein should be maintained at the appropriate salt concentration.

2: Select a suitable surfactant and reducing agent to destroy all non-covalently bound protein complexes and covalently disulfide bonds to form a solution of the respective polypeptide.

3: Try to remove interference molecules such as nucleic acids, polysaccharides, and lipids.

4: Prevent artificial modification of proteins during sample processing. The preparation process should be carried out at low temperature to avoid modification of various enzymes released by cell disruption (recommended to add suitable protease inhibitors)

5: The sample is recommended to be packed into a suitable amount, then freeze-dried or directly stored in a liquid state at -80 ° C, but be careful not to freeze and thaw repeatedly.

Below we detail the detailed steps of the western blot experiment.

1. Preparation:

a water bath, an ultrasonic device, a tissue homogenizer

3. Operation steps:

1) Preparation of cultured cell protein samples:

1: Pyrolysis after trypsin digestion:

When the cells were cultured to a density of about 80%, they were digested with 0.05% trypsin, and the cells were rinsed three times with pre-cooled PBS, collected by centrifugation in a centrifuge tube, and repeatedly blown with 450 μl of the lysate.

2: Direct lysis on the dish:

When the cells were cultured to a density of about 80%, the cells were rinsed three times with pre-cooled PBS, 450 μl of the lysate was added, and the cells were collected by a spatula, transferred to a centrifuge tube with a pipette, and repeatedly blown.

The sample obtained above was finally sonicated with a sonic cell disrupter, 5S during ultrasonication, 10S intermittent time, 100-120W power until the solution was clear and viscous, the treatment was carried out in a water bath, and then centrifuged at 25000g for 1 hour at 4°C. Take the supernatant or ultrasound at maximum intensity for 4 times, each time 15-30S, transfer the sample to 15S in the water bath between each ultrasonic step, and add Laemmli sample buffer (as the protein sample concentration, 1:1 or 1: Mix 2 in a ratio), mix thoroughly, place the sample in a water bath at 100 ° C for 3-5 minutes in a water bath, centrifuge at 10,000 g for 10 minutes, remove the clear solution, and transfer it to another clean tube. At this point, the electrophoresis sample is ready. (The sample can be used immediately or frozen separately, and the sample stored at -20 °C can be kept stable for several months).

2) Preparation of tissue samples:

The surgically removed tissue block was quickly placed in pre-cooled 0.95 normal saline, rinsed several times to clean the surface of the blood, the tissue was weighed and cut into several smaller tissue blocks into the machine or tissue homogenizer. According to the net weight of tissue, the ratio of lysate = 1:10, add the corresponding volume of lysate for homogenization, and collect the supernatant by centrifugation (if there is sticky substance, it can be sonicated. For the specific method, see sample preparation of cell culture, or freeze-dry. After degrading the nucleic acid, the lyophilized protein sample is dissolved in a suitable loading buffer, and after mixing for 3 hours, the protein in the sample is sufficiently dissolved, and it can be collected in 5 minutes and centrifuged at 4 degrees.) Adding Laemmli sample The buffer (depending on the protein sample concentration, mixed in a ratio of 1:1 or 1:2) is vigorously mixed. The sample is heated in a water bath of 100 degrees for 3-5 minutes, centrifuged at 10,000 g for 10 minutes, and the supernatant is taken. Transfer to another clean test tube. At this point, the electrophoresis sample is ready (the sample can be used immediately or frozen, and the sample stored at -20 °C can be kept stable for several months.)

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