Western blot is one of the most frequently performed experiments in molecular biology, biochemistry and immunology. High-quality western blot results can be a perfect plus for a research project.

Western Blot first goes through PAGE (based on molecular size) of separated protein samples, which are transferred to a solid phase carrier (e.g. PVDF), and then the target protein is incubated with a specific antibody. The expression of the protein is reflected by detecting the grayscale of the protein strip bands^[1].

Western blot requires multiple steps such as sample preparation, electrophoresis, membrane transfer, incubation of antibodies and development (e.g. Figure 1).

A well-prepared buffer is the first step to success. The recipes for the various types of buffers involved in western blot are summarized below:

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Once the solution is prepared, it's time to separate the proteins from the sample! To maximize the original structure of the proteins, make sure that all operations are performed on ice!

1. Preparation of cell lysate: Wash cells with pre-cooled PBS on ice. Add pre-chilled lysis buffer at a ratio of 1 mL of lysis buffer per 1x10⁷ cells. Place the cell resuspension at 4 °C for 5-10 minutes with 3-4 vigorous shocks of 30 seconds each to achieve adequate lysis. Centrifuge at 12,000 rpm for 13-15 minutes. Transfer the supernatant to a pre-chilled centrifuge tube on ice and discard the sediment. In the case of tissue lysate preparation, dissect the target tissue quickly and multiple centrifugations are recommended to remove impurities.

2. Preparation of protein sample: Determine the protein concentration by Bicinchoninic Acid Assay (BCA). Remove 50 μg of protein corresponding to the solution into a centrifuge tube, and add 1× SDS loading buffer to level the volume. Boil the cell lysate in the sample buffer for 5-10 min at 100 °C.

After preparing the sample, it is time to run the gel. Choose the appropriate gel according to the size of the protein (Table 2). Then load the protein sample into the SDS-PAGE gel wells, remembering to dot the marker in the well next to the sample. The total protein sample volume of cell lysate or tissue homogenate is usually 20-30 μg, and the length of electrophoresis is usually 1.5 hours, first run at 80 V for 30 minutes to make each sample at the same level, then turn the voltage to 120 V for another hour after the marker starts to separate.

The main methods of membrane transfer are wet transfer and semi-dry transfer. The transfer time should be adjusted accordingly to the protein size. Wet transfer means that the gel and membrane are immersed in a tank filled with transfer buffer during the transfer process, and is suitable for most conventional proteins of various molecular weights. The semi-dry transfer is where the gel and membrane are sandwiched between filter paper pre-wetted with buffer, and the filter paper is in direct contact with the plate electrode. The wet transfer is more widely used and relatively more time efficient, usually requiring only about 10 min, depending on protein size and instrument conditions.

Regardless of whether it is a semi-dry or wet transfer, M has a few common tips for you:

a) Immerse the PVDF membrane in pre-chilled methanol for 5s to activate it.

b) Pay attention to removing the air bubbles between the film and adhesive when transferring the film.

c) Placement order of film transfer materials (Figure 2).

Typically, membranes need to be sealed with sealing buffer for 1 hour at room temperature prior to incubation of primary antibodies, thus allowing proteins or antibodies to effectively adhere to the membrane surface.

Membranes are washed 3 times with PBST for 5 minutes each, followed by incubation of the sealed membranes with diluted primary antibody overnight at 4°C. Incubate the membranes for 2 hours at room temperature with the coupled secondary antibody diluted in a blocking buffer.

After washing with PBST, incubate membranes with ECL Chemiluminescence solution for 1-3 minutes and collect images using Chemiluminescence Imager. Note: Avoid light throughout the entire process after adding ECL chemiluminescence solution.