Antibody Dilution Calculator
Volumes of antibody stock and buffer for a 1:X dilution, a working concentration (ng/mL to mg/mL) or an amount per test, for any number of samples with overage. Adds a secondary antibody, an intermediate dilution when the volume is too small to pipette, a titration series and the cost per run.
How it works
Total volume = volume per sample × samples × (1 + overage) · antibody volume = total ÷ dilution factor · factor = stock ÷ working concentration (C₁V₁ = C₂V₂) · per-test mode: working concentration = µg per test ÷ test volume · final concentration = stock ÷ factor
Antibody datasheets give a ratio (1:1000 for western blots, 1:100–1:1000 for immunofluorescence), a working concentration in µg/mL or ng/mL, or, for flow cytometry, an amount per test. Each reduces to one dilution factor. The total volume divided by that factor is the stock to add, and the rest is buffer. Stock and working concentrations can be entered in mg/mL, µg/mL or ng/mL, and the sample volume in µL or mL. If the stock volume falls below what you can pipette accurately (about 1 µL on most P2 or P10 pipettes), the calculator plans a 1:10 to 1:1000 intermediate dilution first. Under More options you can make a secondary antibody in the same volume and see how many runs a vial gives and what each costs. A titration series at half and double the dilution helps when optimising a new antibody.
Worked example
12 blots × 100 µL + 10% = 1,320 µL at 1:1000 → 1.32 µL of antibody in 1,318.7 µL of buffer. With a 1 mg/mL stock the final concentration is 1 µg/mL and the run uses 1.32 µg; a 100 µL vial covers 75 such runs. In concentration mode, 2 µg/mL from a 1 mg/mL stock is 1:500, so the same volume takes 2.64 µL. For flow cytometry at 0.25 µg per 100 µL test from a 0.2 mg/mL stock, the working concentration is 2.5 µg/mL, a 1:80 dilution.
Sources: Thermo Fisher — Western blot antibody dilution calculator
Frequently asked questions
Can I reuse diluted primary antibody?
Often, for 2–3 uses within a couple of weeks at 4 °C with 0.02% sodium azide (not for HRP secondaries — azide inhibits HRP).
What diluent?
Typically 5% BSA or milk in TBST for blots, blocking buffer for staining. Milk masks phospho-epitopes — use BSA for phospho-antibodies.
How do I convert a 1:1000 dilution into µg/mL?
Divide the stock concentration by the dilution factor: 1 mg/mL (1,000 µg/mL) at 1:1000 is 1 µg/mL. Enter the stock concentration and the calculator shows the final concentration for any ratio.
What if the antibody volume is less than 1 µL?
Make an intermediate dilution. Dilute the stock 1:10 or 1:100 in buffer, then use 10 or 100 times the volume of that. The calculator picks the intermediate and gives both steps once the volume drops below your pipetting limit.
How should I titrate a new antibody?
Test a series around the datasheet value, for example a quarter, half, one, two and four times the dilution, on the same samples. Pick the dilution with the best signal-to-background, not the strongest signal. The titration table gives the volumes for each.
Related calculators
For teaching and planning — verify against your lab protocol and instrument documentation.