How do I make a 1 to 1000 antibody dilution?

Divide your final volume by 1000 to get the antibody volume, then make the rest up with diluent. For 1 millilitre of working solution that is 1 microlitre of antibody plus 999 microlitres of buffer. For 5 millilitres it is 5 microlitres of antibody plus 4995 microlitres. The ratio always refers to the final total volume, not the volume of diluent alone.

What does a 1 to 500 dilution actually mean?

It means one part antibody in 500 parts total, so 1 part antibody plus 499 parts diluent. This is the convention in almost every antibody datasheet. It is worth being careful because a small number of older protocols write 1 to 500 meaning one part in 500 parts of diluent, which gives a total of 501 parts. The difference is under a quarter of a percent, so it rarely matters in practice, but the total-volume reading is the standard one.

How do I convert a working concentration into a dilution factor?

Divide the stock concentration by the working concentration you want, keeping both in the same units. A 1 milligram per millilitre stock is 1000 micrograms per millilitre, so getting to a 2 micrograms per millilitre working solution needs a factor of 1000 divided by 2, which is 500, or a 1 to 500 dilution. Datasheets often give a stock concentration and a recommended working concentration rather than a ratio, so this conversion comes up constantly.

What if the antibody volume is too small to pipette?

Do the dilution in two steps. Anything below about 1 microlitre cannot be pipetted accurately, and even 1 to 2 microlitres carries large percentage error on most pipettes. Split the total factor across two steps, for example 1 to 5000 becomes 1 to 100 followed by 1 to 50, since 100 multiplied by 50 gives 5000. Make a small volume of the intermediate dilution, then dilute that. Both steps then use volumes you can measure reliably.

What dilutions are typical for each application?

As rough starting points: primary antibodies for Western blot are often 1 to 1000, secondaries 1 to 5000 or higher, immunohistochemistry and immunofluorescence around 1 to 200 to 1 to 500, flow cytometry near 1 to 100, and ELISA detection antibodies 1 to 5000. These are only starting points. The datasheet for your specific antibody and lot should always take priority, and optimal dilution is usually found by titration.

Antibody Dilution Calculator with Pipetting Check

Dilution 1 :

Final volume

Final volume units

Smallest volume you can pipette (µL)

Antibody

—

add this much stock

Diluent

—

buffer to make up the rest

How antibody dilutions work

A dilution written as 1:500 means one part antibody in 500 parts total, not one part in 500 parts of diluent. So the antibody volume is simply the final volume divided by the dilution factor, and everything else is buffer. For 5 mL at 1:500 that is 5000 µL / 500 = 10 µL of antibody plus 4990 µL of diluent.

When a datasheet gives a working concentration instead of a ratio, convert first: divide the stock concentration by the working concentration, in matching units. A 1 mg/mL stock is 1000 µg/mL, so reaching 2 µg/mL needs 1000/2 = a 1:500 dilution. From there it is the same calculation.

Why very high dilutions need two steps

A 1:5000 dilution in 200 µL needs 0.04 µL of antibody. No pipette measures that, and even 1–2 µL carries a large percentage error on most pipettes. The fix is to split the factor across two steps, because dilution factors multiply:

1:100 then 1:50 = 100 × 50 = 1:5000 step 1 1 µL antibody + 99 µL diluent → 100 µL of 1:100 step 2 4 µL of that + 196 µL diluent → 200 µL of 1:5000

Both volumes are now comfortably pipettable, and the final concentration is identical. The calculator above flags this automatically and picks a sensible split whenever the direct volume falls below what you can measure.

Typical starting dilutions

Application	Typical range
Western blot, primary	1:500 – 1:2000
Western blot, secondary (HRP)	1:2000 – 1:10000
Immunohistochemistry (IHC)	1:100 – 1:500
Immunofluorescence (IF/ICC)	1:200 – 1:1000
Flow cytometry	1:50 – 1:200
ELISA detection	1:1000 – 1:10000

These are starting points only. The datasheet for your specific antibody and lot always takes priority, and the optimal dilution is normally found by titrating a small range around the recommendation.

FAQ

How do I make a 1:1000 dilution?
Final volume ÷ 1000 is the antibody volume. For 1 mL: 1 µL antibody + 999 µL buffer.

Does 1:500 include the antibody?
Yes, it means 1 part in 500 total, so 1 part antibody + 499 parts diluent.

How do I turn a working concentration into a ratio?
Divide stock by working concentration in the same units. 1 mg/mL (1000 µg/mL) to 2 µg/mL = 1:500.

What if the volume is too small to pipette?
Split it into two steps whose factors multiply to the total, for example 1:100 then 1:50 for 1:5000.

Which dilution should I start at?
Follow the datasheet, then titrate around it. The table above gives rough ranges per application.