III. Reagents and Methods
2. Extraction, characterization and quantification of milk proteins
Milk proteins were extracted from different sources and subsequently characterized and quantified using quantification using SDS-PAGE electrophoresis and Bradford assay, respectively. As discussed previously, most milk proteins can be divided into caseins and whey proteins. The quantity of each protein varies in different milk products, such as liquid pasteurized milk, powdered milk and specialized hypoallergenic milk formulas.
The extraction, characterization and quantification of these proteins allowed for their use in in vitro digestion experiments, and later in bioaccessibility and bioavailability assays.
2.1. Optimization of the extraction of milk proteins
The extraction of milk proteins was achieved following with the method used in (Vincent et al., 2016), with slight alterations. An extraction buffer containing (6M Ureia;
75mM NaCl; 10mM TrisHCl; 10mM DTT; 0,05% SDS; pH 8) was prepared, and the pH of the solution was adjusted to 8. Skim milk and powdered milk samples were split into 3 aliquots each, into 15mL falcon tubes. For skimmed milk samples, 5mL of extraction buffer were added to 5 ml of skimmed milk (containing approximately 0.16g of protein, in accordance with the information present in the nutrition label), for a total volume of 10ml.
For the powdered milk samples, 0,55g of powdered milk (also containing 0.16g of protein) were added to 5 mL of extraction, for a total volume of 5 mL (Figure 10). All solution where then vortex for 1 minute, followed by a 60 min incubation at 30ºC, with constant agitation. After the incubation period, all solutions were centrifuged at 13000rpm for 5min, at room temperature. The resulting pellet was discarded and the supernatant containing the proteins was collected.
Figure 10 – Schematic representation of the milk protein extraction process.
The resulting supernatant was then dialyzed with a 12-14kDa cut-off during 4 hours at room temperature, with water changes every 1h, and then overnight at room temperature, all with constant agitation.
Finally, the dialyzed solutions were frozen at -20ºC and lyophilized. The resulting dry product was then stored at -20ºC.
2.2 . Protein quantification of milk proteins in milk protein extract
The QuickStartTM Bradford protein assay, by BioRad was used to quantify the protein content in milk extracts following the manufacturer instructions. Briefly, solutions of 2g/mL of bovine serum albumin (BSA) and commercial casein were used as protein standards. Both protein standards were diluted in different wells of a 96-welled plate, with a solution contain 6M urea and 0,5 TrisHCl at pH 6.8. Solutions were prepared according to Table 2, in triplicate:
Table 2 – Volumes and concentrations of used in the preparation of casein and BSA calibration curves
Final [Protein]
(μg/ml)
Standard Volume (μl)
Source of Standard
Diluent Volume (μl)
2000 20 2mg/ml stock 0
1500 30 2mg/ml stock 10
1000 20 2mg/ml stock 20
750 20 1500ug/ml
solution 20
500 20 1000ug/ml
solution 20
250 20 500ug/ml
solution 20
125 20 250ug/ml
solution 20
0 - - 20
The protein extracts obtained during the extraction process where then dissolved in the same solution used to dilute the protein samples. The resulting solutions had a concentration of 1mg/ml. 20μl of each sample was then pipetted into separate wells, with 6 replicates per sample.
After this, 250ul of 1× dye reagent was added to each well. Samples were then incubated in the dark for 15min, at room temperature. Samples absorbance was read at 595nm and used to quantify the protein content of each sample. The resulting data was analysed using the GraphPad Prism software.
2.3. Optimization of the characterization of milk proteins
In other to characterize the protein extract obtained in the last chapter, an SDS-PAGE gel electrophoresis was performed. The samples were loaded into a 4% acrylamide for stacking gel and 12% acrylamide for the running gel.
Protein extracts and milk protein standards were dissolved in a solution composed of 6M urea and 0,5M TrisHCl, at pH 6.8. The resulting solutions had a protein concentration of 1,25ug/ml. 36ul of the prepared solutions were added to 10μl of 5× sample buffer and 4ul off a 10mM DTT solution, resulting a protein concentration of 0,9ug/ml and a DTT concentration of 0,8M. All the solutions were then incubated at 95ºC for 5 min.
10μl of the previous solutions were then loaded into the agarose gels, resulting in 9ug of protein per well. 0,25mA were applied until the samples reached the running gel, after which the current applied was 0.30mA.
2.4. Scale-up of the protein extraction process
After the characterization and quantification of milk proteins in skimmed milk protein extract, the previous method was used to perform an extraction on a larger scale. The extraction protocol previously described was thus used to extract 200ml of skimmed milk.
In other to characterize the protein extract obtained in the last chapter, an SDS-PAGE gel electrophoresis was performed. The samples were loaded into a 4% acrylamide for stacking gel and 12% acrylamide for the running gel.
Protein extracts, powdered milk and milk protein standards were dissolved in a solution composed of 6M urea and 0,5M TrisHCl, at pH 6.8. The resulting solutions had a protein concentration of 1,25ug/ml. 36ul of the prepared solutions were added to 10μl of 5× sample buffer and 4ul off a 10mM DTT solution, resulting a protein concentration of 0,9ug/ml and a DTT concentration of 0,8M. Skimmed milk was also diluted in the same solution until a final concentration of 1,25ug/ml of proteins. All the solutions were then incubated at 95ºC for 5 min.
10μl of the previous solutions were then loaded into the agarose gels, resulting in 9ug of protein per well. 0,25mA were applied until the samples reached the running gel, after which the current applied was 0.30mA.