Question

How to ensure the label from heavy and light protein was 100% effective? How do you verify the proteins are 100%?

Answer 1

To determine dye-to-protein molar ratio, the extinction molar coefficient (ε) of the unlabeled protein must be known. Each kind of fluorescent dye molecule absorbs maximally at a particular wavelength to an extent described by its extinction coefficient (ε´). Amax is the absorbance (A) of a dye solution measured at the wavelength maximum (λmax). Together, the Amax and ε´ may be used to calculate the molar concentration of dye in a sample.

Calculate molarity of the protein:

• ε = protein molar extinction coefficient
• Amax = Absorbance of dye measure at wavelength max.
• CF = Correction factor
• Dilution factor = Amount

Protein Concentration (M) = ((A280 -(Amax x CF)) ÷ ε) X Dilution factor

Calculate the degree of labeling:

• ε' = dye molar extinction coefficient

Moles dye per mole protein = (Amax of labeled protein ÷ (ε' x protein conc (M))) x dilution factor

If the protein has mixture of heavy and light ammino acids. Efficiency of labelling count for both heavy and light ammino acid containing protein.

Stable isotope labeling of amino acids (SILAC) labeling: The SILAC peptide count ratio analysis (SPeCtRAmethod relies on MS spectra. To validate the SPeCtRA method, we have analyzed samples constructed with known ratios of protein abundance

Arginine or lysine are heavy amino acid and identified in the heavy search (SCheavy) whereas Proteins : unlabeled (light).

To determine the efficiency of labeling cell line. Percent labeling of each protein was calculated as:

%Heavy = SCheavy/(SCheavy+SClight)∗100