"<h4>1.Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on ice throughout).</h4>"+

"<h4>2.Add PBS and use a cell scraper to dislodge the cells. Pipette the mixture into microcentrifuge tubes.</h4>"+

"<h4>3.Centrifuge at 1500 RPM for 5 minutes and discard the supernatant.</h4>"+

"<h4>4.Add 180 μL of ice cold cell lysis buffer with 20 μL fresh protease inhibitor cocktail. (Tip: If protein concentration is not high enough at the end, it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail).</h4>"+

"<h4>5.Incubate for 30 minutes on ice, and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C.</h4>"+

"<h4>6.Transfer supernatant (or protein mix) to a fresh tube and store on ice or frozen at -20°C or -80°C.</h4>"+

"<h4>7.Measure the concentration of protein using a spectrophotometer.</h4><br>"+

"<h4>1.</h4>"+

"<h4>2.Add 5 μL sample buffer to the sample, and make the volume in each lane equalized using double distilled H2O (dd H2O). Mix well. (Tip: Total volume of 15 μL per lane is suggested).</h4>"+

"<h4>3.Heat the samples with dry plate for 5 minutes at 100°C.</h4><br>"+

"<h4>1.After preparing the 10% stacking gel solution, assemble the rack for gel solidification. (Tip: 10% AP and TEMED solidify the solution; therefore, both gels can be prepared at the same time, if the abovementioned reagents are not added until the end).</h4>"+

"<h4>2.Add stacking gel solution carefully until the level is equal to the green bar holding the glass plates. Add H2O to the top. Wait for 15–30 minutes until the gel turning solidified. (Tip: Using a suction pipette can make the process of adding the gel to the glass plate easier).</h4>"+

"<h4>3.Overlay the stacking gel with the separating gel, after removing the water. (Tip: It is better to tilt the apparatus and use a paper towel to remove the water).</h4>"+

"<h4>4.Insert the comb, ensuring that there are no air bubbles.</h4>"+

"<h4>5.Wait until the gel is solidified. (Tip: Solidification can be easily checked by leaving some gel solution in a tube).</h4><br>"+

"<h4>1.Pour the running buffer into the electrophorator</h4>"+

"<h4>2.Place gel inside the electrophorator and connect to a power supply. (Tip: When connecting to the power source always connect red to red, and black to black).</h4>"+

"<h4>3.Make sure buffer covers the gel completely, and remove the comb carefully.</h4>"+

"<h4>4.Load marker (6 μL) followed by samples (15 μL) in to each well</h4>"+

"<h4>5.Run the gel with low voltage (60 V) for separating gel; use higher voltage (140 V) for stacking gel.</h4>"+

"<h4>6.Run the gel for approximately an hour, or until the dye front runs off the bottom of the gel.</h4><br>"+

"<h4>1.Cut 6 filter sheets to fit the measurement of the gel, and one polyvinylidene fluoride (PDVF) membrane with the same dimensions.</h4>"+

"<h4>2.Wet the sponge and filter paper in transfer buffer, and wet the PDVF membrane in methanol.</h4>"+

"<h4>3.Separate glass plates and retrieve the gel.</h4>"+

"<h4>4.Create a transfer sandwich as follows:</h4>"+

"<h4>(Tip: Ensure there are no air bubbles between the gel and PVDF membrane, and squeeze out extra liquid).</h4>"+

"<h4>5.Place electrodes on top of the sandwich, ensuring that the PVDF membrane is between the gel and a positive electrode. Parameter setting of the transmembrane: A=[Area of Gel*3/4]/1000, constant current mode.</h4>"+

"<h4>6.Transfer for 7 minutes. </h4>"+

"<h4>7.Wash the membrane with ddH2O for 5min. Do this 3 times. (Tip: Shaking the membrane slowly.)</h4><br>"+

"<h4>1.Block the membrane with 3% bovine serum albumin ( BSA) at 37℃，350r/min shaking, for 2 hour. </h4>"+

"<h4>2.Wash the PVDF membrane with TBST Buffer for 10min. Do this 3 times. (Tip: Shaking the membrane slowly.)</h4>"+

"<h4>3.Add primary antibody in 5% bovine serum albumin ( BSA) and incubate overnight in 4°C on a shaker.</h4>"+

"<h4>4.Wash the membrane with TBST for 5 minutes. Do this 3 times. (Tip: All washing and antibody incubation steps should be done on a shaker at room temperature to ensure even agitation).</h4>"+

"<h4>5.Add secondary antibody in 5% skim milk in TBST, and incubate for 1 hour.</h4>"+

"<h4>6.Wash the membrane with TBST for 5 minutes. Do this 3 times</h4>"+

"<h4>7.Prepare ECL mix (following the proportion of solution A and B provided by the manufacturer). Incubate the membrane for 1–2 minutes. (Tip: Use a 1000 μL pipette to ensure that ECL covers the top and bottom of the membrane).</h4>"+

"<h4>8.isualize the result in the dark room. (Tip: If the background is too strong, reduce exposure time).</h4><br>"+

"<h4>5.Sterilize by filtration or autoclaving</h4><br>";