Antibody dilution calculator
Microlitres of antibody and diluent for a 1:X dilution, or for a target concentration in µg/mL.
Microlitres of antibody and diluent for a 1:X dilution, or for a target concentration in µg/mL.
Antibody datasheets give the working dilution as a ratio such as 1:1000. That is one part of antibody stock in 1000 parts of final solution, so the volume of antibody is the final volume divided by the dilution factor, and the diluent makes up the rest.
Worked example. A primary antibody is used at 1:1000 and the blot needs 10 mL. Antibody volume = 10 mL ÷ 1000 = 0.01 mL = 10 µL, added to 9.99 mL of blocking buffer. If the stock is 1 mg/mL, the working concentration is 1000 µg/mL ÷ 1000 = 1 µg/mL.
When the datasheet gives a working concentration instead of a ratio, which is usual for flow cytometry, ELISA coating and purified monoclonals, the calculation is C₁V₁ = C₂V₂ with the stock converted to the same unit first (1 mg/mL = 1000 µg/mL).
Worked example. Stock 1 mg/mL, target 2 µg/mL, final volume 5 mL: 2 × 5 ÷ 1000 = 0.01 mL =10 µL of antibody plus 4.99 mL of diluent. That is the same as a 1:500 dilution. Any other reagent can be diluted the same way with the generaldilution calculator.
| Application | Ratio | Concentration |
|---|---|---|
| Western blot, primary | 1:500 – 1:5000 | 0.1 – 2 µg/mL |
| Western blot, HRP secondary | 1:2000 – 1:20,000 | 0.05 – 0.5 µg/mL |
| Immunofluorescence / ICC | 1:100 – 1:1000 | 1 – 10 µg/mL |
| Immunohistochemistry | 1:50 – 1:500 | 2 – 20 µg/mL |
| ELISA, detection | 1:500 – 1:5000 | 0.2 – 2 µg/mL |
| Flow cytometry | 1:50 – 1:200 | 0.5 – 5 µg/mL, or µg per 10⁶ cells |
These are starting points. The datasheet value for your antibody and lot takes priority, and a new antibody is best titrated over a few two-fold or five-fold steps around it. Too much antibody raises background and non-specific bands; too little loses signal.
A 1:10,000 dilution in 2 mL needs 0.2 µL of antibody, which no pipette delivers reliably. There are two fixes. Make a larger volume, if antibody is not limiting. Or dilute in two steps: first 1:100 (2 µL into 198 µL), then 1:100 again (20 µL of the intermediate into 1.98 mL). The factors multiply, 100 × 100 = 10,000. The calculator proposes such a scheme whenever the antibody volume falls below 1 µL. For longer series see theserial dilution planner.
Divide the final volume by 1000 to get the antibody volume, and make up the rest with diluent. For 10 mL of a 1:1000 dilution, add 10 µL of antibody to 9.99 mL of blocking buffer. In practice adding 10 µL to 10 mL is the same within pipetting error.
In antibody datasheets and almost all molecular biology protocols, 1:1000 means one part antibody in 1000 parts total, a 1000-fold dilution. The other reading, 1 + 1000, differs by 0.1% at this dilution, which does not matter. The distinction only becomes important for low dilutions such as 1:2 or 1:5.
Divide the stock concentration by the dilution factor. A 1 mg/mL antibody is 1000 µg/mL, so at 1:1000 it is 1 µg/mL, and at 1:5000 it is 0.2 µg/mL. The reverse works too: stock concentration divided by the target concentration gives the dilution factor.
Volumes under about 1 µL cannot be pipetted accurately. Either make more working solution than you need, or make an intermediate dilution first, for example 1:100 in the same diluent, and dilute that the rest of the way. The calculator suggests a two-step scheme when this happens.
Follow the datasheet. For western blots it is usually the blocking buffer, 5% non-fat milk or BSA in TBST; for phospho-specific antibodies BSA is preferred because milk contains phosphoproteins. For immunofluorescence and IHC it is typically PBS with 1% BSA or normal serum. Diluted primary antibody in buffer with 0.02% sodium azide can often be reused several times when stored at 4 °C.