Amenaza De Los Nuevos Entrantes

3.5.4.1Description of the technique

Microfluidic chip electrophoresis (MF-electrophoresis) is a miniaturised electrophoresis technique that uses capillary electrophoresis and works on the same principle as traditional SDS-PAGE. The proteins are moved across the chip in microfluidic channels that are filled with a polymer matrix and proteins are separated according to their molecular weight. The procedure includes similar steps as for the SDS-PAGE method, such as protein sample preparation, sample loading in the wells of the chip, electrophoretic separation, protein staining and destaining and detection and quantification of the proteins by integration. These steps are detailed below. The application of MF-electrophoresis for milk protein separation has been described in detail and compared with SDS-PAGE by Anema (2009a).

Two main advantages can be pointed out compared with SDS-PAGE: (i) MF-electrophoresis shortens the time of analysis and (ii) no gel making procedure is involved and the staining/destaining procedure is immediate. Therefore, a set of 10 samples can be analysed for protein content and composition within 30 min. It also has a larger linear range of detection than traditional SDS-PAGE, so samples containing low levels of proteins can be analysed with more accuracy and with further dilution needed.

3.5.4.2Procedure

The MF-electrophoresis was performed using an Agilent 2100 Bioanalyzer system and the associated Protein 80 kit (Agilent Technologies, Waldbronn, Germany). The kit contained a gel matrix solution, a concentrated protein dye solution, and a concentrated ladder solution containing proteins of known molecular mass.

The following solutions were prepared:

Protein ladder

The diluted protein ladder was prepared by adding 84 μL of Milli-Q water to 6 μL of the protein ladder supplied in the kit.

Gel dye and destaining solution

Gel dye and destaining solution were prepared according to the protocols supplied with the chips. The gel matrix provided in the kit was added in an Eppendorf tube equipped with a disposable filter and centrifuged for 15 min at 2500 × g. The filtered solution was used as it is for destaining solution. 25 μL of concentrated dye was added to the filtered solution to make up the gel dye solution.

Sample buffer

Two hundred and fifty microlitres of Milli Q water, 62.5 mL of 0.5 M Tris-HCl buffer, pH 6.8, and 50 mL of 10% (w/v) SDS (50 mL) were mixed together, then 1 mL of 0.3 mg/mL lactoferrin was added to 150 mL of the sample buffer before further use. Lactoferrin was used as the upper marker in the integration step while SDS was the lower marker.

The protein samples were diluted in sample buffer in 1 mL Eppendorf tubes to obtain a final protein concentration comprised between 0.01% and 0.1%. 5% β-mercaptoethanol was

added and the samples were heated at 100 °C for 5 min, as explained for traditional SDS- PAGE.

Figure 3.2 shows the layout of a MF-electrophoresis chip. A chip contains 16 wells, of which three are for the gel dye (G1 to G3, G5), one is for the destaining solution, one is for the protein ladder and ten are for the protein samples. A chip was filled first with 12 μL of gel dye in G1, and air pressure was applied on the well with a syringe for 1 min mounted on a supplied loading station. The rest of the wells were then loaded with 12 μL of gel dye in G2, G3, G4, 12 μL of destaining solution in well DS, 6 μL of ladder in well L and 6 μL of samples in wells S1 to S10. Once loaded, the chip was inserted to the machine and the lid was closed. The electrophoresis was started and lasted for ~ 30 min.

Figure 3.2: Layout of the wells and channels in a typical microfluidic electrophoresis chip. The separations channel (A), position of destaining (B) and position of the detection window (C) are highlighted with arrows. Source: Anema (2009a).

3.5.4.3Interpretation of the results

A typical elution profile obtained for reduced SM by MF-electrophoresis is shown in Figure 3.3. From left to right of the elution time profile, the milk proteins appear in the order α- lactalbumin, β-lactoglobulin, β-casein, αS-casein (αS1 +αS2), κ-casein. Lower molecular weight

According to the molecular weight of the different proteins, the peak of κ-casein should have been on the left side of the peak of β-casein, as κ-casein has the lowest molecular weight of the caseins. A faster elution time corresponds to a longer distance of migration on the SDS- PAGE gels. On the SDS-PAGE gels, κ-casein migrated further than αS-casein and β-casein

(band lower on the gel). This anomalous behaviour of κ-casein in microfluidic chip electrophoresis has been reported before and has been attributed to potential artefacts affecting the elution position of κ-casein in the technique (Anema, 2009a). However, it does not affect the quantification of κ-casein. The Agilent 2100 expert software (Agilent Technologies) automatically integrated the protein peaks, but the integration usually had to be refined manually. The reported values for protein quantification are the peak areas.

Figure 3.3: Typical elution profile obtained from MF-electrophoresis technique for reduced skim milk. The peaks were (a) SDS lower marker, (b) α-lactalbumin (c) β- lactoglobulin, (d) β-casein, (e) αS-casein (αS1 +αS2), (f) κ-casein, (g) lactoferrin upper marker.

Skim milk was diluted in sample buffer at 1:20 ratio.

From the elution profile given in Figure 3.3, a computer generated gel image is given by the software. Figure 3.4 shows a typical gel image of skim milk analysed by MF-electrophoresis, at two different dilution ratios in sample buffer. The protein bands appear from bottom to top of the gel in order of increasing elution times, in the order α-lactalbumin, β- lactoglobulin, β-casein, αS-casein (αS1 +αS2), κ-casein. The order of the protein bands on the

Elution time (s) 10 20 30 40 50

In

te

n

s

it

y

b

c

d

e

f

g

a

gel is similar than that obtained on a traditional SDS-PAGE gel, and proportional to their molecular weight, except for κ-casein, that has a different migration position on MF- electrophoresis gels, where the κ-casein band is located on top of the other casein bands, compared with on traditional SDS-PAGE gels, where the κ-casein band is located under the β-casein band.

Figure 3.4: Computer-generated gel image from the elution profile of reduced skim milk obtained by MF-electrophoresis. Skim milk was diluted in sample buffer at the ratios 1 to 10 (lane 1) and 1 to 20 (lane 2); the axis on the left shows the elution times.