Sin-Hong Pan Using 8 × 10⁵ iPSC-derived cardiomyocytes generally yields around 20–40 µg of total protein, which is sufficient for detecting moderately to highly expressed proteins by Western blot, but may be inadequate for low-abundance targets. If you're targeting a low-expression protein, it's recommended to increase the cell number to 1.5–2 × 10⁶ to obtain at least 30–50 µg of protein for better detection.

Hello Sin-Hong Pan

A single mammalian cell typically contains approximately 50-200 picograms of protein. If your target protein is a low-abundance protein, 40ug of total protein per lane may be sufficient as a good starting point, but optimization may be crucial. If the target protein is highly abundant, a lower protein load (like 10-20ug of total protein per lane) might be sufficient to generate a strong signal.

So, considering 200pg of protein per cell, you will require 2 x 10^5 cells to get a total protein of 40ug to be loaded per lane on the gel for detecting low-abundant protein in Western Blot. Similarly, you will require 1 x 10^5 cells to get a total protein of 20ug to be loaded per lane on the gel for detecting highly abundant protein in Western Blot. So, the cell count of 8 × 10^5 – 1 x 10^6 should suffice for your experiment.

Please note that the quality and dilution of your antibody are critical for detecting low-abundance proteins. Ensure you are using an antibody validated for Western Blotting and that is specific for your target protein. If you are using a highly specific antibody, you may be able to detect your protein with a lower protein load.

Best,

5x10^5 cells are totally OK for WB, I will direct add 1x loading buffer without centrifuge.