Alpha lactalbumin

An experiment was conducted to validate the quantification techniques. Additional bulk samples containing 15 µg of yeast proteins (four independent replicates) were supplemented with purified enzymes at different concentrations: insulin (Sigma, ref. I5500, Saint Louis, MO, USA) at 51.9 fmol; alpha-lactalbumin (Sigma, ref. L5385, Saint Louis, MO, USA) at 108.6 fmol; myoglobin (Sigma, ref. M0630, Saint Louis, MO, USA) at 181.8 fmol; and ribonuclease A (Sigma, ref. R5500, Saint Louis, MO, USA) at 342.6 fmol. The samples underwent in-gel digestion as described above. The extracted tryptic peptides were vacuum dried and resuspended in 75 μL of loading buffer. Then, 1.5 µL of digested UPS2 (424 ng/µL) was spiked into 7.5 µL of the digested peptides (from the yeast proteins and purified enzymes). The expected and observed protein abundances for the samples were compared for the different quantification techniques.

Cytochrome c (from bovine heart), lysozyme (from chicken egg white), alpha lactalbumin (from bovine milk), DL-homocysteine thiolactone hydrochloride (HTL) and 8-Anilinonaphthalene-1-sulphonic acid were purchased from Sigma-Aldrich chemical Co. Potassium chloride, di-potassium hydrogen phosphate and potassium di-hydrogen phosphate were purchased from Merck, India. Dithiobis(2-nitrobenzoic acid), the Ellman's reagent was also purchased from Sigma-Aldrich chemical Co. Guanidium hydrochloride (Gdmcl) was purchased from M.P. Biomedicals. Unless otherwise stated, all chemicals were used without further purification and double distilled water was used as aqueous phase.

Trizma Base, EDTA, Sodium Hydroxide, Sodium dodecyl sulphate, Ethidium bromide, Imidazole, Tween-20, Tween-80, Sodium chloride, Potassium chloride, Glycerol, Acrylamide, bis-acrylamide, Ammonium persulphate, TEMED, Glycine, β-mercaptoethanol, Hydrogen peroxide (H₂O₂), Dithiothreitol (DTT), Horseradish peroxidase (HRP), 5,5′-dithiobis(2-nitrobenzoate) (DTNB), Bovine carbonic anhydrase, and Alpha-lactalbumin were purchased from Sigma Aldrich Pvt. Ltd.
Luria Bertani Medium and Luria Bertani Agar (Difco), PBS tablets (Biobasic), Ni–NTA resin (Qiagen GmbH, Germany), DNA markers (GeneDireX Inc., USA), Precision dual colour Protein marker (Bio-Rad). Ampicillin and Kanamycin were purchased from MP Biomedicals.
Primary (Monoclonal Anti-Prdx6, clone 3A10-2A11 antibody produced in mouse, WH0009588M1) and secondary (Anti-mouse IgG (Fab Specific)-Peroxidase antibody produced in goat, A3682) to detect full length human Prdx6 protein was procured from Sigma-Aldrich.