3.4. Western Blotting and Detection

For the detection of ERK1/2 phosphorylation, load 10–20 μL of the sample on SDS-PAGE gel and resolve the proteins by running the gel at 100–120 V. As the molecular weights of ERK1 and ERK2 are very close to each other (42kDa and 44kDa), it is important to run the gel long enough to visualize a clear separation. If prestained protein marker is being used, separation of different bands can be used as a proxy to roughly estimate the duration for running the gel.

Once proteins in the lysate are resolved by SDS-PAGE, rinse the gel in Milli-Q water to get rid of residual SDS as it can interfere with the protein transfer onto PVDF membrane followed by an additional wash in the transfer buffer. In the meantime, activate the PVDF membrane by incubating it in methanol for 2–5 min and then set-up the protein transfer on the membrane using semidry transfer assembly.

Once the protein transfer is done, incubate the PVDF membrane in the blocking buffer (5% BSA in 1 × TBST) for 1h at room-temperature on a rocking platform to block any nonspecific antibody binding sites on the membrane. The composition of 1 × TBST is—19 mM Tris-Cl, 137 mM NaCl, 0.1% Tween 20, pH 7.5. Subsequently, take the membrane out of the blocking buffer and incubate it with antiphospho-ERK1/2 antibody solution (1:5000–10,000 dilution) prepared in 5% BSA in TBST for 8–12h at 4°C.

After primary antibody incubation, wash the membrane three times (5–10 min each) with 1 × TBST under moderate shaking. Incubate the membrane with HRP-conjugated antirabbit secondary antibody (1,5000–10,000 dilution) for 1–2 h at room-temperature with moderate shaking. Subsequently, wash the membrane three times (5–10 min each) with 1 × TBST under moderate shaking and then add 1–2 mL of ECL substrate to cover the entire membrane. Capture the image with CCD camera attached to the ChemiDoc imaging system or using conventional X-ray film.

A key step in the Western blot based ERK assay is to probe the samples for total ERK1/2 using the same membrane as used for detecting phospho-ERK1/2 signal. For this, incubate the membrane (after phosph-ERK1/2 signal is captured) with 15–30 mL of stripping buffer (Glycine 200 mM, 0.1% SDS (w/v), 1% Tween 20 (v/v), β-Mercaptoethanol 0.08% (v/v) and pH adjusted to 2.2 with HCL) for 15–30 min followed by washing in 1 × TBST (three times, 5–10 min each) and blocking of nonspecific binding sites using 5% BSA in 1 × TBST for 1 h at room-temperature on a rocking platform. Subsequently, incubate the membrane with antitotal-ERK1/2 antibody (1:5000–10,000) for 2 h at room-temperature or 8–12 h at 4°C. Capture the signal following the protocol as described above for the phospho-ERK1/2.

Quantify the intensity for phospho-ERK1/2 and total-ERK1/2 bands using densitometry, plot the normalized data as phospho-ERK/total-ERK, and analyze using appropriate statistical measures.

Note

Transfer buffer should be prepared fresh with a final composition of 48 mM Tris-Cl, 39 mM Glycine, 20% Methanol, pH 9.2. Alternatively, a stock of 10 × transfer buffer can be prepared and stored without the methanol, and methanol can be added freshly just before setting up the transfer. Methanol helps in getting rid of any residual SDS bound to the gel, and enhances the ability of the proteins to bind the PVDF membrane, and therefore, it is a critical to activate the membrane with methanol prior to setting-up the protein transfer.

Nonfat dry milk powder should not be used for blocking the nonspecific binding sites on PVDF membrane as casein present in milk powder may result in high background signal. Membrane should be washed carefully and thoroughly to minimize the background signal, and the incubation with HRP-coupled secondary antibody should preferably be carried in dark. pH of the buffers should be carefully measured just before the experiment.

Phospho-ERK1/2 antibody may be used more than once, and therefore, it can be stored at 4°C with 0.02% NaN₃ for a month or so.