Sample Preparation

Preparation of samples for Western blotting (WB) generally involves four main steps:

Sample isolation — Components, organelles, cells, or tissues must be isolated from the specific source of interest. Additional requirements depend on the sample type. For adherent cells, simple trypsinization may be sufficient, whereas heterogeneous tissues may require removal of unwanted components such as lipids or collagen. Eliminating these contaminants can improve overall experimental quality.

Lysis — Cells, organelles, or tissue samples are treated with an appropriate lysis buffer, and proteins are released through mechanical disruption. Selection of a suitable lysis strategy is critical for obtaining high-quality results. In some cases, such as cell culture supernatants, lysis may not be required; however, protein abundance may be low, making sample loading more challenging. Under these conditions, protein precipitation or concentration methods (e.g., TCA precipitation or protein concentrators using centrifugal filter units) may be necessary.

Protein quantification — This step ensures sufficient protein input and consistent loading across samples. It is important to note that certain protein quantification methods may be incompatible with components in the lysis buffer, such as detergents or EGTA. Verifying compatibility in advance is essential for selecting an appropriate assay.

Reduction and denaturation — Samples are typically treated with reagents to disrupt higher-order protein structures (secondary and tertiary structures) and linearize proteins, ensuring separation based strictly on molecular weight. Sample buffers commonly contain DTT or β-mercaptoethanol to reduce disulfide bonds, as well as SDS to form denatured protein–SDS complexes. In addition, samples are usually briefly boiled or at least heated prior to loading.

After protein extraction, the concentration can be determined by BCA method or Bradford method to ensure stable and controllable loading amount.

After determining the protein concentration of each lysate, take equal quality lysate and add 4xSDS Sample Loading Buffer.

After mixing, boil at 95-100°C for 5-10 minutes to reduce and denature the sample. The lysate can be aliquoted and stored at -20°C for later use.

Note: For some membrane proteins, transmembrane proteins, and even nuclear proteins (Rb protein), high-temperature boiling may cause aggregation to form multimers or "ghost bands", leading to electrophoresis band shift or loss.

SDS-PAGE Electrophoresis

Tips

• 1) Ammonium persulfate decomposes slowly and needs to be prepared freshly in time;
• 2) During electrophoresis, proteins not only diffuse and separate from top to bottom, but also diffuse and separate to both sides, so add appropriate amount of 1x loading buffer (or irrelevant protein sample) to empty sample wells to prevent "smiling" bands;
• 3) Under the premise of not affecting the detection signal, try to load less sample, the resulting bands will be sharper;
• 4) Do not overheat during electrophoresis, overheating will cause band dispersion or even protein degradation;
• 5) Prepared gels can be stored in a 4°C refrigerator for about 2-4 weeks (prevent drying, prevent freezing and thawing).

Transfer (Take Wet Transfer as an Example)

Tips

• 1) Try to clean the sponge thoroughly after each transfer; filter paper should not be reused as much as possible;
• 2) Transfer time needs to be optimized according to protein size, reference constant voltage for wet transfer of 20~250kD: 75V, 80-90 minutes;
• 3) Transfer buffer can be used at most 2 times to avoid excessive heat generation leading to protein degradation;
• 4) Wet transfer is recommended for large molecular weight proteins, semi-dry transfer may not necessarily improve transfer efficiency even if the time is extended (and may even lead to degradation);
• The transfer of protein Marker and target protein is not completely equivalent (common in semi-dry transfer), especially for high molecular weight proteins.

Antibody Incubation

Development

Tips

• The choice of developing solution is also very important, the laboratory should at least have two sensitivity levels of ECL. It is recommended to prepare the developing solution fresh, and when trying a new target for the first time, it is best to try with low sensitivity ECL first, and then change to high sensitivity ECL if it is weak.
• 1) When shooting with an imager, do not recommend using the machine's auto-exposure mode; it is recommended to shoot 5 or 10 images within a fixed time, and select the best image later according to the actual situation;
• 2) Imagers can generally adjust parameters such as image zoom, resolution/sensitivity.

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