Antibody Dilution Calculator
Use this antibody dilution calculator to prepare a working dilution such as 1:500 or 1:1000. Enter the dilution factor and the total volume you need — it returns the antibody and diluent to pipette.
Enter the dilution factor X and the total volume to get the antibody and diluent to pipette.
How to calculate an antibody dilution
An antibody dilution written 1 : X means one part antibody in X parts total working solution. So the antibody volume is the total divided by the dilution factor:
The diluent is whatever your protocol specifies — PBS, TBST, blocking buffer or antibody dilution buffer with BSA. The working concentration is the stock concentration divided by X.
Worked example
For a 1:1000 dilution in 5 mL (5000 µL): antibody = 5000 ÷ 1000 = 5 µL, plus 4995 µL of diluent. At a 1 mg/mL stock that is a 1 µg/mL working concentration.
Typical dilution ranges by application
Starting points vary a lot by assay — always check the antibody's datasheet, but these are common ranges:
| Application | Typical dilution |
|---|---|
| Western blot — primary antibody | 1:500 – 1:5,000 |
| Western blot — secondary (HRP) | 1:2,000 – 1:10,000 |
| Immunohistochemistry (IHC) | 1:50 – 1:500 |
| Immunofluorescence (IF) | 1:100 – 1:1,000 |
| ELISA (capture / detection) | 1:1,000 – 1:10,000 |
| Flow cytometry | 1:20 – 1:200 |
A note on conventions
This tool treats 1:X as "1 part in X total", the standard for antibodies and stains. If your method means 1 part stock to X parts diluent instead, use the dilution ratio calculator, which adds the parts together (X + 1 total). To seed cells at a target density, see the cell dilution calculator.
Frequently asked questions
›How do I make a 1:500 antibody dilution?
A 1:500 dilution means 1 part antibody in 500 parts total, so antibody volume = total ÷ 500 and diluent = total − antibody. For 5 mL (5000 µL) of working solution: 5000 ÷ 500 = 10 µL of antibody plus 4990 µL of diluent (buffer, BSA or blocking solution).
›How do I calculate a working antibody concentration?
Divide the stock concentration by the dilution factor. A 1 mg/mL stock at 1:1000 gives a working concentration of 1 mg/mL ÷ 1000 = 1 µg/mL. The dilution factor is the "X" in 1:X.
›Does 1:X mean 1 part in X, or 1 part to X parts?
For antibody dilutions the near-universal convention is "1 part in X total" — so 1:1000 is antibody diluted to one-thousandth, using antibody = total ÷ X. That is what this calculator uses. A dilution ratio calculator instead reads 1:X as 1 part stock to X parts diluent (X + 1 total), which is slightly different — check which your protocol means.
›Is this antibody dilution calculator free?
Yes. It runs entirely in your browser, needs no sign-up, and never sends your numbers to a server.
›Why does the same antibody need such different dilutions across assays?
Sensitivity and background tolerance differ a lot by technique — IHC/IF tissue backgrounds tolerate less antibody than a clean ELISA plate, so working dilutions vary 10-100× across applications. Always start from the datasheet's suggested range and titrate from there.
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