How to Reconstitute a Lyophilized Protein or Peptide

The formula

volume to add = mass in the vial ÷ target concentration

Keep the units matched: mg ÷ mg/mL gives mL, and µg ÷ µg/µL gives µL.

Examples

• 5 mg of peptide at 2 mg/mL: 5 ÷ 2 = 2.5 mL.
• 100 µg of antibody at 0.5 mg/mL (0.5 µg/µL): 100 ÷ 0.5 = 200 µL.
• 1 mg of a 1,500 g/mol peptide at 1 mM: 0.001 g ÷ 1,500 g/mol = 0.667 µmol, and 0.667 µmol ÷ 1 mmol/L = 667 µL.

Check how much is really in the vial

A synthetic peptide's powder is not all peptide. Counterions such as trifluoroacetate and bound water often make up 10% to 30% of the weight, so the certificate of analysis may give a net peptide content of, say, 80%. Multiply the gross mass by that fraction before calculating: 5 mg at 80% is 4 mg of peptide. For proteins, check the amount on the datasheet, and after dissolving, confirm the concentration by absorbance at 280 nm.

Choosing the solvent

• Follow the datasheet first. Most proteins and antibodies go back into water or the buffer they were freeze-dried from.
• Peptides with many basic residues (K, R, H) dissolve better in a little dilute acetic acid; acidic ones (D, E) in a little dilute ammonium bicarbonate.
• Very hydrophobic peptides may need a small volume of DMSO first, then slow dilution into buffer. Avoid DMSO for peptides with cysteine or methionine, which it can oxidize.

Steps

1. Let the vial reach room temperature before opening, so moisture does not condense on the powder.
2. Spin it briefly so all the powder is at the bottom.
3. Add the solvent slowly down the side of the vial.
4. Swirl or roll gently and leave it for 15 to 30 minutes. Do not vortex proteins hard, which can denature and foam them.
5. If it stays cloudy, a brief bath sonication can help peptides. Particles that remain are not dissolved, so spin them down and measure the concentration of the supernatant.

Storing it

Split the solution into single-use aliquots and freeze them at −20 °C or −80 °C, because each freeze–thaw cycle can cost activity. Dilute proteins below about 0.1 mg/mL stick to tube walls; a carrier protein such as 0.1% BSA prevents that when it will not interfere with the experiment.

Calculators

The Reconstitution Calculator gives the volume for a target concentration, or the concentration from a volume, in mass or molar units. Check the result with the Protein Concentration Calculator (A280), and plan working dilutions with the Stock Solution Calculator.