How It Works
This calculator works in 4 steps.
Enter the secondary dilution
Type the ratio from the protocol or datasheet.
Enter the working volume
Type the total detection volume.
Check the transfer volume
The calculator flags antibody volumes under 1 µL and offers a predilution.
Read the volumes
You get the antibody and buffer volumes. With a predilution, you get both steps.
The Formula
The formula divides the working volume by the ratio term and splits high ratios into 2 stages.
V_secondary = V_total / XX = X_pre × X_finalV_pre = V_total / X_final- X:
- Total ratio term
- X_pre:
- Predilution factor, such as 10
- X_final:
- Second-stage factor
- V_total:
- Working volume
1:20,000 in 15 mL needs 0.75 µL secondary. With a 1:10 predilution, add 7.5 µL of the predilution to 15 mL buffer.
Conversion Table
The conversion table gives secondary and buffer volumes for 6 ratios and volumes.
| Secondary dilution | Working volume | Secondary antibody | Buffer |
|---|---|---|---|
| 1:2,000 | 10 mL | 5 µL | 9,995 µL |
| 1:5,000 | 10 mL | 2 µL | 9,998 µL |
| 1:10,000 | 10 mL | 1 µL | 9,999 µL |
| 1:20,000 | 15 mL | 0.75 µL | 14,999 µL |
| 1:500 | 1 mL | 2 µL | 998 µL |
| 1:1,000 | 0.5 mL | 0.5 µL | 499.5 µL |
What Is the Secondary Antibody Dilution Calculator?
The Secondary Antibody Dilution Calculator is a tool for the detection step. It turns the ratio you pick into antibody and buffer volumes. The secondary binds the primary. It carries the label you detect, such as HRP or a dye that glows.
Secondaries are often used more dilute than primaries. HRP conjugates for chemiluminescent westerns appear on datasheets in the 1:2,000 to 1:20,000 range. At 1:20,000, 10 mL needs only 0.5 µL. That is too small to pipette well. So the calculator flags any volume under 1 µL and suggests a 1:10 predilution in buffer.
Common Uses
There are 5 main uses of this calculator.
HRP westerns
Users dilute HRP secondaries for ECL detection.
Fluorescent westerns
Users dilute near-IR secondaries for scanners.
Immunofluorescence
Cell labs dilute dye-tagged secondaries for slides.
ELISA detection
Teams dilute enzyme-tagged antibodies for the read step.
Predilution planning
Labs prepare 1:10 secondary predilutions for repeated use.