Many peptides are insoluble in water. The Hydrophobic Peptide Assay Kit (Catalog number HPEP200) is designed for concentration measurement of hydrophobic peptides in DMSO. The assay is based on increase of fluorescence at 535 nm of the dye C33 in the presence of peptides. The assay sensitivity is in the range between 0.02 and 0.2 mg/ml depending on the nature of peptides. The assay is not compatible with amino acids, thiol compounds such as DTT or metal chelators such as EDTA.
The Hydrophobic Peptide Assay Kit (Catalog number HPEP200) includes 1 ml of 10 x C33 dye for 200 assays using 96-well plates. Cuvettes may also be used for the assay.
ASSAY PROTOCOL
The sample volume is 0.15 ml and the final volume is 0.2 ml in the 96-wellplate assay format.
STANDARD CURVE
1. Dilute 50 µl of 10 x C33 dye 10 fold with ethanol to make 500 µl of 1 x C33 dye.
2. Prepare 150 µl of peptide solutions in DMSO in the wells of a black 96-well plate with a two[1]fold serial dilution from 0.5 mg/ml to zero in DMSO.
3. Mix 50 µl of 1 x C33 dye with 150 µl of the peptide solutions for 10 min.
4. Read the fluorescence at 535 nm (excitation at 485 nm).
5. Plot the fluorescence intensity Fc and the peptide concentration [Peptide] to generate the linear standard curve.
Fc = a [Peptide] + b
Where the Fc values are from experimental data, the a and b values are from the linear fitting between the Fc values and the peptide concentrations.
UNKNOWN SAMPLES
Follow the same procedure to measure the fluorescence intensity Fc values from the unknown samples. Calculate the peptide concentrations in the unknown samples using the Fc values from the unknown samples and the a and b values from the standard curve.