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The Isolation Of Milk Whey

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Day 1: Isolation of Whey The isolation of milk whey began with 10 mL of nonfat milk which had been centrifuged at 16,000 x g for 45 minutes in a refrigerated centrifuge. The top layer***. 10 mL of the nonfat milk were then pipetted into a small glass beaker. The pH of the nonfat milk was slowly adjusted from a pH of 6.60 to a pH of 4.60 through the dropwise addition of 0.5 M and 0.05 M HCl. The coagulated solution was heated to approximately 40°C for 30 minutes while being constantly stirred.
While the solution was being heated, 100 mL of elution buffer was made using 0.02 M Tris, 0.5 M NaCl, and 0.02 M imidazole with a final pH of 7.0. 100 mL of binding buffer and 100 mL of stripping buffer were obtained as well. The binding buffer had …show more content…

The previous wash and collection of fractions was done again using the elution buffer. All 10 fractions collected were analyzed using UV spectra at 280 nm using the *** as the blank.
Day 3: Protein Characterization Using SDS-PAGE Gel Electrophoresis and BCA Assay A crude whey sample and a purified *-lactalbumin sample were prepared for SDS-PAGE gel electrophoresis by mixing 20 µL of each sample with µL of the reducing gel buffer in Eppendorf tubes. These two samples were then boiled for 5 minutes and then allowed to cool to room temperature. A precast gel was inserted into the gel running apparatus and the comb was removed. Consequently, a 10x stock solution of tank buffer was diluted tenfold and added to middle of the chamber until it covered the gel. Then, * µL of crude whey sample was added to well 10 and the purified sample was pipetted into well 4. A standard MW ladder was loaded into well 1 of the gel. The remaining buffer was poured into the bottom the tank. The top was placed and the gel was then run at a constant 150 V for ***. After running the gel, it was removed from the apparatus and was rinsed several times with water. To fix the gel, it was rocked in a container with 50 mL of water and 1 mL of glutaraldehyde for 10 minutes. The glutaraldehyde mixture was decanted and enough gel code stain to cover the gel was added to the container. The container was

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