Contexto de aplicación de la experiencia “los viernes al campo”

Figure 3.1 shows an initial SDS-PAGE gel of samples from the adhesion assay between mucin coated Sepharose beads and partially in vitro gastric digested whey. Control samples (“Whey,

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Figure 3.12: Adhesion assay between mucin-beads and digested whey

Reducing SDS-PAGE gel of MUC2 covered Sepharose beads after incubation with digest mix (acidified whey); EtOH and LiCl wash solutions prepared with PBS 5.5. Gel separated at 130 V for 2 hr and stained with CBB G250 overnight. Bold font indicates samples that contain Sepharose beads (i.e. shows adhering proteins). NC: Negative control.

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pH 2” to “NC, pure MUC2 bead”) show good protein band resolution. Protein profiles in “Whey, pH 2” and “Time 0 control” are comparable to the profile of whey observed in Figure 3.1. Control sample “1 min digest” shows that most proteines were hydrolysed and only bands for pepsin (35 kDa), monomeric β-LG (18 kDa), little α-LA (14 kDa) and peptides (<10 kDa) can be observed. Samples from the adhesion assay show that no protein or peptide fraction bound completely to the mucin coated beads (“Removed SN”) and that most proteins and peptides were washed off the mucin coated beads during the first wash with PBS. Only a faint band of β-LG was observed from “MUC2 bead A”, suggesting that protein adhesion to the beads was very limited. Further, no protein bands could be observed in the collected wash supernatants. To resolve this issue, the volume loaded onto the gels was increased. 12% and 15% acrylamide SDS-PAGE gels (1 mm width) were cast instead of using precast gels. This allowed doubling the sample volume from 30 μl to 60 μl. Figure 3.13 shows a representative 12% acrylamide gel. Overall, this SDS-PAGE gel is comparable to the one in Figure 3.12 with regards to band pattern. In particular, the band intensity in the wash solutions was comparable with the one in pre-cast gels (e.g. Figure 3.12). However, the resolution, especially in the higher molecular weight region, was very poor. To get a better quality gel for this part, 15% acrylamide gels were prepared. These resulted in a poor resolution for the lower molecular weight proteins (data not shown). Overall the qualities of self-cast gels did not meet the requirements for clear band correlation between samples throughout the whole molecular weight range of interest (10 to 150 kDa) and these gels were discontinued. As the increase of sample volume did not result in stronger bands, an attempt was made to increase the protein concentration within the samples by precipitating and re-dissolving in less volume. EtOH precipitation and chloroform-MeOH precipitation were assayed and chloroform-MeOH precipitation was determined best suited for the samples as it resulted in gels with more distinct bands (Figure 3.14 and Figure 3.15). Figure 3.14 shows removed whey and wash solutions. Figure 3.15 has the same gel layout as previous SDS-PAGE gels (e.g. Figure 3.12). In both gels, protein and peptide fractions are enriched due to precipitation which increased the load onto SDS-PAGE gels from 30 µl untreated wash solution up to 2 x 150 µl solution precipitate, a 10-

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Figure 3.13: Adhesion assay between mucin-beads and digested whey; 12% acrylamide SDS-PAGE gel

Reducing SDS-PAGE gel, 12% acrylamide, of MUC2 covered Sepharose beads after incubation with 1 min digested whey. No EtOH-amine controls for beads A, B and C. Double volume of sample compared with pre-cast gels loaded onto gels in order to increase band intensities in wash solutions. Gel separated at 25 V overnight and stained with CBB G250 overnight. Bold font indicates samples that contain Sepharose beads (i.e. shows adhering proteins). NC: Negative control.

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Figure 3.14: Adhesion assay between mucin-beads and digested whey, concentrated by EtOH- precipitation

Reducing SDS-PAGE gel of complete adhesion assay with EP of 100 μl wash solutions. Gel separated at 130 V for 2 hr and stained with CBB G250 overnight. NC: Negative control. EP: Ethanol precipitation. 55 kDa 45 kDa 34 kDa 17 kDa 7 kDa 4 kDa 210 kDa 78 kDa 105 kDa

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Figure 3.15: Adhesion assay between mucin-beads and digested whey, concentrated by CMP

Reducing SDS-PAGE gel of MUC2 covered Sepharose beads after incubation with digest mix (whey); Full adhesion assay with CMP of 100 μl wash solutions. Gel separated at 130 V for 2 hr and stained with CBB G250 overnight. Bold font indicates samples that contain Sepharose beads (i.e. shows adhering proteins). NC: Negative control. CMP: chloroform-MeOH precipitation.

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Figure 3.16: Scheme of the final adhesion protocol including sample preparation

Pellet A and pellet B in the figure are named “bead A” and “bead B” in samples with mucin coated Sepahorse beads, and “pellet A” and “pellet B” in samples containing bacterial cells or IEC. CMP: chloroform-MeOH precipitation. Precipitated samples are indicated by smaller tubes in the figure.

1. Substrate (binding buffer) and surface component mixed

2. Mix incubated for a set time at 37°C, if possible under agitation

After incubation, supernatant removed

3. Pellet wash with protein free binding buffer Supernatant collected

Pellet A subsampled

4. Pellet wash with PBS 5.5 Supernatant collected

5. Pellet wash with 25% EtOH Supernatant collected

6. Pellet wash with 2.5 M LiCl

Supernatant collected Pellet B sampled Wash PBS Pellet A Samples Protocol Wash LiCl Pellet B Wash PBS 5.5 Wash EtOH

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fold increase of protein load onto pre-cast gels. EtOH precipitation caused more streaking and also appeared to interfere with the EtOH wash (Figure 3.14 “Wash, EtOH2, EP 100 µl”). Thus CMP was considerd the more practicable method for further assays. Despite precipitation, only few bands at 18 kDa were observed in the wash solutions (Figure 3.15).