How is the sample volume calculated?

Sample volume equals target mass divided by measured concentration. To load 20 µg from a 2 µg/µL lysate you pipette 10 µL. Lower-concentration samples need more volume, so the calculator also checks each fits in the well.

How much loading buffer do I add?

Loading buffer is added to reach 1× in the final volume. For a 4× buffer in a 20 µL well you add 5 µL (one quarter), since 4× diluted four-fold becomes 1×. The remaining volume after sample and buffer is made up with water.

What if a sample is too dilute to fit the well?

If the required lysate volume plus the loading buffer already exceeds the final well volume, you cannot reach the target mass at that well size. The calculator flags the sample so you can concentrate it, lower the target, or use a larger well.

Why normalise to equal protein per lane?

Western blot band intensity is only comparable across lanes if each carries the same total protein. Equal loading lets you attribute differences in band signal to the target protein's abundance rather than to how much lysate you happened to load.

Should I still run a loading control?

Yes. Even with careful normalisation, assays and transfers vary. A housekeeping loading control such as GAPDH, β-actin, or total-protein stain confirms equal loading and lets you correct the signal if needed.

Western Blot Sample Preparation Calculator

A western blot only quantifies protein abundance fairly if every lane carries the same total protein mass. This calculator takes each lysate’s measured concentration and works out exactly how much sample, water, and loading buffer to pipette so all lanes are normalised to one target mass in one final volume.

How it works

For each sample, the lysate volume needed is the target mass over the measured concentration, the loading buffer is a fixed fraction set by its strength, and water fills the rest:

sample_vol = target_mass / concentration
buffer_vol = final_vol / buffer_strength      (e.g. final/4 for 4× buffer)
water_vol  = final_vol − sample_vol − buffer_vol

If sample_vol + buffer_vol already exceeds the final well volume, the target mass cannot be reached at that well size and the sample is flagged.

Example and tips

To load 20 µg into a 20 µL well with 4× buffer: a 2.5 µg/µL lysate needs 8 µL of sample, 5 µL of 4× buffer, and 7 µL of water. A weak 0.5 µg/µL lysate would need 40 µL of sample alone, which won’t fit — concentrate it or lower the target. Keep the target mass realistic for your detection: 10–30 µg of total protein per lane is typical for an abundant target, while low-abundance proteins may need more. Always pair normalisation with a loading control to confirm it worked.