ANALYTICAL METHODS · PRACTICAL GUIDE
Serial Peptide Dilution: Follow Each Tube and Plate Position
Follow a dilution series tube by tube, including the volume left after each transfer.
A row of tubes labeled 100, 50 and 25 µM looks straightforward. The difficulty is remembering which solution moves where, and how much remains. We will follow one twofold dilution series from the first tube to the plate.
Why equal volumes halve the concentration
Start with 100 µL at 100 µM and add 100 µL of suitable diluent. The final volume is 200 µL. If no material is lost, the amount of peptide stays the same while the volume doubles, so the concentration becomes 50 µM.
100 µM × 100 µL ÷ 200 µL = 50 µM
Follow the next four tubes
Keep enough 100 µM solution in tube 1. Put 100 µL of diluent in each of tubes 2–5. Transfer 100 µL from tube 1 into tube 2 and mix; then transfer 100 µL from tube 2 into tube 3 and mix. Continue in that order.
The concentrations are 100, 50, 25, 12.5 and 6.25 µM. Mix before taking the aliquot for the next tube, so the transferred liquid represents the newly diluted solution.
Count the liquid left in each tube
Tube 2 contains 200 µL after mixing, but only 100 µL after you transfer half to tube 3. The same applies to the later transfer tubes. The final tube remains at 200 µL if you do not remove an aliquot.
That difference matters when you plan how many wells each tube can fill. The first tube’s remaining volume also depends on how much starting stock you prepared.
A working solution may be diluted again in the well
If a 50 µM working solution is mixed with an equal volume of another solution containing no peptide, the final concentration is 25 µM. The concentration on the tube is not automatically the concentration in the well.
Draw a plate map showing concentration, replicates and control wells. This keeps the dilution calculation connected to the actual layout.
Check one more step
See the answer
12.5 µM. The final volume is 160 µL. If you then remove 80 µL, the remaining solution is still 12.5 µM, but only 80 µL remains.
The calculation assumes the peptide stays dissolved and is not lost to the container. If recovery matters at low concentrations, investigate that separately.
Using a dilution plan with KPV or LL-37
When planning KPV or LL-37 dilutions, check the stock concentration and solvent before starting. Keep the dilution table with the product form and lot details so a later batch can be compared under the same planned conditions.
Discuss material and document requirements with NHD · Continue with the analytical guide