Jaesung Lee -

1. Yes, normalizing sample loading based on total protein concentration (e.g., using a BCA assay) is generally more appropriate than normalizing by cell number, especially in your case where it can induce apoptosis.

2. Non-specific bands in Western blotting are common but can raise question during publication (generally speaking). In your case the band HER2 dimer, phosphorylated HER2 or some interacting partner. One suggestion is you can perform an immunoprecipitation (IP) with the HER2 antibody followed by WB to see if this band co-precipitates with HER2.

3. Thinner, sharper bands will improve the visual quality and credibility of your data. As you are already planning to reduce the protein conc. that can help, aslo try using a 10% gel than a gradient gel.

Thank you for your help.

I hadn’t considered immunoprecipitation, but it seems like a valuable approach.

Since this is my first time doing Western blotting, I was a bit lost, but your advice really helped me find direction.

Much appreciated!

Q1. Use your BCA to approximate protein loading and your reference gene (GAPDH in this case) to normalize.

Q2. Both bands are likely related (they are affected by your siRNA). The additional band could be due to dimerization, PTM, etc Did you use reducing and denaturing conditions during the 95 °C treatment?

Do not IP as it will introduce more variability.

Q3. Your bands are thick because your gel is probably overloaded. Consider loading one-third or even less of your protein extracts.

Q1 -Yes it is more appropriate to normalize loading based on total protein concentration measured by BCA (or similar) rather than cell number, as lysis efficiency and protein yield can vary between samples, especially if ASOs induce apoptosis. For Q2, a faint non-specific band above HER2 that also decreases with ASO treatment is not uncommon; reviewers may ask about it, but you can address it by clearly labeling it as non-specific in your figure legend and, if possible, confirming HER2 knockdown with an orthogonal method (e.g., qPCR or a different antibody). The fact that it parallels HER2 reduction might simply indicate that it’s another protein affected by your treatment, but it’s worth noting in your discussion. For Q3, your bands do look slightly thick, which can be due to overloading protein, high antibody concentration, or gel percentage. Reducing the protein load (e.g., to 10–15 µg), optimizing gel percentage, and adjusting antibody dilution can help achieve sharper bands within an acceptable width.