Protein
Western blot antibody dilution
Scale antibody and diluent volumes for a western blot across several membranes. Enter the incubation volume per membrane, the number of membranes and an overage, and the primary and secondary dilutions; the tool gives the antibody and diluent for each step.
| Step | Dilution | Antibody | Diluent |
|---|
The antibody volume is the total incubation volume divided by the dilution fold; the diluent is the rest. The total carries the membrane count and overage so a shared trough or bag does not run short. Optimal dilutions are antibody- and lot-specific — take them from the datasheet and refine by titration.
Formula
antibody = total ÷ dilution; total = volume per membrane × membranes × (1 + overage)
- volume per membrane
- the incubation volume one membrane needs
- dilution
- the fold dilution of the antibody, entered as 1:X
- overage
- a margin so a shared trough or bag does not run short
Worked example
A 1:1000 primary across two membranes
- 5 ml per membrane
- 2 membranes, 10% overage
- primary 1:1000
- Total: 5 × 2 × 1.10 is 11 ml.
- Antibody: 11 ml ÷ 1000 is 11 µl.
- Diluent: the remaining 10.989 ml.
Answer 11 µl of primary antibody in ~11 ml of diluent
Common mistakes
- Trusting the datasheet dilution blindly. It is a starting point; the best working dilution depends on your lysate, exposure and lot, and is worth titrating once.
- Forgetting the overage. Troughs and bags leave dead volume; without a margin the last membrane sits in too little antibody.
- Reusing diluted secondary too many times. Each reuse drops the effective titre; note how many times a diluted antibody has been used.
Frequently asked
Should I include the buffer recipe here?
The buffers tool holds TBST, transfer and running buffers and scales them. This tool covers the antibody dilution, which the buffers tool does not.
Does the diluent matter?
Yes — blocking agent in the diluent affects background. This tool sizes the volume; which blocker to use is a method choice for your antibody.
Can I add more than two antibodies?
The table covers a primary and a secondary, the usual pair. For a multiplex, run the tool once per antibody with its own dilution.