Western blotting is a qualitative method for detecting the expression of target proteins in samples by using the specific reaction characteristics of antigens and antibodies. It is the most commonly used method for detecting gene protein expression and is widely used in basic scientific research.
1. Experimental principle
Under the action of an electric field, total proteins are separated according to molecular weight by polyacrylamide gel electrophoresis, and then transferred to a solid phase carrier (nitrocellulose membrane or PVDF membrane). After the carrier membrane is incubated with antibodies that specifically recognize specific antigens, it is specifically bound to a secondary antibody labeled with horseradish peroxidase. Finally, horseradish peroxidase substrate is added for color development (darkroom exposure or chemiluminescence imaging), and the expression of the target protein is analyzed by combining the optical density of the color band.
2. Experimental steps
2.1 Total protein extraction
2.1.1 Tissue samples: Wash tissue blocks 2-3 times with pre-cooled PBS to remove blood stains, weigh, cut into small pieces and place in a homogenizer tube, add homogenizer beads, add 100 μL tissue lysis buffer for every 10 mg of tissue (add protease inhibitors within a few minutes before use), set the homogenization program for homogenization. After homogenization, incubate on ice for 30 min, shake once every 5 min to ensure complete lysis of the tissue. Centrifuge at 12000 rpm, 4 ℃, for 10 min, collect the supernatant, which is the total tissue protein solution.
2.1.2 Cell samples: Discard the culture medium in the culture plate, add an appropriate amount of 4 ℃ pre-cooled PBS to wash the cells 2-3 times, and discard the washing solution. Add an appropriate amount of lysis buffer (containing PMSF and phosphatase inhibitors) to the culture plate according to the instructions, use a pipette to mix, and incubate on ice for 20 min. After the lysis is completed, use a pipette to transfer the lysate to a 2 mL centrifuge tube and centrifuge at 4 °C, 12000 rpm for 10 min. Collect the supernatant, use the BCA method to determine the concentration of the protein solution, and homogenize the protein concentration of each group of samples to adjust to the same concentration. Store the supernatant at -20 °C or -80 °C for later use.
2.2 Gel electrophoresis
2.2.1 Gel preparation: Prepare the separation gel according to the molecular weight of the protein to be analyzed, and inject it into the two layers of glass plates continuously and smoothly along the upper left corner of the glass plate. After the separation gel is polymerized and solidified, pour the concentrated gel solution and immediately insert the gel comb. It can be used after full polymerization.
2.2.2 Protein denaturation: Take out the cell lysate and add 5× Loading Buffer in proportion, mix well, boil in boiling water for 5 min, and immediately place on ice.
2.2.3 Sample electrophoresis: Place the electrophoresis tank correctly, add electrophoresis solution, load the sample, and first perform electrophoresis at a constant voltage of 80 V for 25 min. After the bromophenol blue indicator protein is compressed, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue is about to reach the bottom of the gel.
2.3 Electrotransfer
Remove the gel glass plate from the electrophoresis tank, cut off the unused part, cut the solid phase membrane of the same size as the gel according to the size of the gel, and then soak it in the electrotransfer solution for activation; at the same time, cut 2 pieces of filter paper and soak them in the transfer buffer. Place them on the transfer instrument in the order of cathode → filter paper → gel → solid phase membrane → filter paper → anode, and press out bubbles. Turn on the power, select the constant current mode, calculate the transfer current according to 1.5 mA/cm2 gel, and transfer for 45 min.
2.4 Immune reaction
2.4.1 After washing the membrane with TBST for 5 min, transfer it to a 5% skim milk powder blocking solution prepared with TBST and block it on a shaker at room temperature for 2 h.
2.4.2 Dilute the primary antibody to an appropriate concentration with antibody diluent, remove the membrane from the blocking solution, put it into a new antibody incubation bag, add the primary antibody solution, squeeze out the bubbles, seal it, and incubate it at 4 ℃ overnight.
2.4.3 Take out the membrane the next day and wash it 3 times with TBST on a shaker at room temperature for 5 min each time.
2.4.4 Prepare the corresponding species secondary antibody dilution according to the primary antibody and incubate it with the membrane for 1 h.
2.4.5 Transfer the secondary antibody dilution and wash it 3 times with TBST on a shaker at room temperature for 5 min each time.
2.4.6 The ratio of color development solution A and solution B is 1:1, chemiluminescence, collect photos, and save the original pictures.
3. Result examples
The results reflect different samples, and the protein expression can be clearly seen under the same treatment and detection conditions (the right picture is the control internal reference GAPDH).
An example of high and low quality western blot images using infrared fluorescence. WM793 melanoma cells were treated with DMSO (CTL) or 2 μM of the MEK inhibitor PD0325901 (MEKi) for 18 h. Rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) and mouse anti-total ERK1/2 were used. The first Western blot panel is an overlay of the fluorescence signals of phosphorylated ERK1/2 (800nm channel - green) and total ERK1/2 (700nm channel - red) detected simultaneously. The second and third panels are black and white images of the single channel fluorescence images of phosphorylated and total ERK1/2.
References
[1] G.J. Anderson, C.M. Cipolla, R.T. Kennedy, Western blotting using capillary.electrophoresis, Anal. Chem. 83 (2011) 1350–1355.
[2] A.J. Hughes, A.E. Herr, Microfluidic western blotting, Proc. Natl. Acad. Sci. U. S. A. 109 (2013) 21450–21455.
[3] S.Q. Tia, M. He, D. Kim, A.E. Herr, Multianalyte on-chip native western blotting,Anal. Chem. 83 (2011) 3581–3588.
[4] S. Jin, G.J. Anderson, R.T. Kennedy, Western blotting using microchip electrophoresis interfaced to a protein capture membrane, Anal. Chem. 85 (2013) 6073–6079.
[5] A.J. Hughes, D.P. Spelke, Z.C. Xu, et al., Single-cell western blotting, Nat. Methods 11 (2014) 749–755.