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Antibody dilution calculator

Calculate antibody stock and diluent volumes for a 1:N dilution, including sample count and preparation allowance, with worked examples and pipetting checks.

Use this calculator when a validated protocol specifies a dilution such as 1:500. The final volume includes both antibody stock and diluent; do not add the stock on top of that volume.

Prepare a working dilution

For 1:500, enter 500. This is a dilution factor, not an antibody concentration.
Choose enough working solution to cover the specimen. For 1 mL, enter 1000.
Preparing several samples or allowing for transfer losses?

Prepare one batch only for samples using the same antibody dilution and diluent. Allowing for transfer losses adds extra solution at the same dilution.

Use a separate calculation for each dilution in a titration series.
Optional extra volume for pipetting or reservoir losses. Leave at 0 if none is needed.
Enter the dilution and final volume, then calculate. Volumes are in µL.

How the dilution is calculated

A 1:N dilution means one part antibody stock in N parts of final working solution, not one part stock plus N parts diluent.

  • Batch final volume = volume per sample × number of samples × (1 + allowance / 100).
  • Antibody stock volume = batch final volume / dilution denominator.
  • Diluent volume = batch final volume − antibody stock volume.

For 1:500 in 1000 µL, use 2 µL antibody stock + 998 µL diluent. For 1:200, four 100 µL samples and a 10% allowance, prepare 440 µL in total: 2.2 µL stock + 437.8 µL diluent. The allowance increases the amount prepared, not the antibody concentration.

At 1:1, the whole final volume is stock and the diluent volume is zero: there is no dilution.

When the stock volume is too small to pipette reliably

For stock volumes below 1 µL, check whether your pipette and tips can dispense that amount reliably. Accuracy depends on the equipment, liquid, and technique; the two decimal places shown in the result do not indicate pipetting precision.

A larger batch or an intermediate dilution can make the stock volume easier to measure. Check that the antibody is stable in the chosen diluent and follow its handling and storage instructions. For example, 1:1000 in 100 µL would require 0.1 µL of original stock. The following calculation uses two dilution steps:

  1. Prepare a 1:10 intermediate: 5 µL original stock + 45 µL compatible diluent, making 50 µL.
  2. The remaining dilution factor is 1000 / 10 = 100. Use 1 µL of that intermediate + 99 µL diluent for a 100 µL final working solution.
  3. The two dilution factors multiply: 10 × 100 = 1000. Do not dilute the intermediate 1:1000 again, which would give a final dilution of 1:10,000.

Mix each dilution appropriately and check reagent compatibility and stability. If 1 µL is outside the reliable range of your equipment, plan a larger batch or a different validated intermediate; do not round 0.1 µL up to 1 µL of original stock.

Choosing the antibody dilution

Choose the dilution, diluent, incubation time, and temperature from the antibody datasheet and protocols for the same application, then check them with a titration and appropriate controls. The calculator gives the volumes for that dilution. Prepare each condition in a titration series separately.

If a protocol specifies a mass concentration rather than a ratio, the stock concentration must be known. For a 1:N dilution, working concentration = stock concentration / N, using the same concentration units. Two antibody lots with different stock concentrations will not necessarily give the same working concentration at the same ratio. For concentration-based calculations, use the C₁V₁ = C₂V₂ calculator with consistent units.

Follow the staining protocol for cultured cells, frozen sections, or FFPE sections. Compare titration results with the appropriate controls, and record the dilution and volumes on the experiment checklist. If staining has high background, check the unstained and secondary-only controls before changing the antibody concentration.