The Western Blot technique is a well-established method used for detecting proteins in homogenised tissue samples. The transfer on a cellulose membrane allows other substances to gain access to the proteins and, if applicable, interact with them. To identify the protein of interest, a primary antibody is added, recognising and binding the specific epitope. Subsequently secondary antibodies, which are
Gene Amplification efficiency (%)
R2 Slope y-Intercept
TBP 104.2 0.998 39.14 -3.225
PPIA 98.6 0.996 40.64 -3.355
GLUT1 97.3 0.998 40.65 -3.389
GLUT2 96.2 0.998 39.89 -3.416
SGLT1 100.8 0.993 39.48 -3.304
IL-4 102.9 0.999 40.10 -3.255
IL-13 95.7 0.998 41.21 -3.429
HIF1α 101.0 0.998 39.18 -3.298
Stat6 113.5 0.988 39.70 -3.036
PepT1 92.9 0.997 39.80 -3.506
Table 3. Parameter of quantitative real-time PCR
labelled with luminol-based substrates, are added and produce a chemiluminescent signal based on the oxidation of luminol. This permits indirect detection.
2.6.1. Western Blot – Preparation of cytosol, crude and apical membranes
Before a Western Blot analysis can be performed, tissue preparation must be conducted. Respective preparation depends on the location of the target protein. ASCT1, PepT1, SGLT1 and its phosphorylated form pSGLT1 are located in the apical membrane, whereas GLUT2 and Na+/K+-ATPase are placed in the basolateral membrane. Hif-1α is found in the cytosol.
Subcellular fractioning was used to divide different subcellular compartments in order to enrich apical membrane proteins and crude membranes for detecting basolateral proteins. At the outset, 1.5 g of sampled tissue was incubated with 13.5 mL homogenization buffer (2 mM TRIS base, 50 mM mannitol, pH of 7.1) until it thawed. Homogenisation with a Potter tissue grinder was followed by centrifugation at 2,000 g for 15 min at 4 °C, and the resulting supernatant was transferred into a beaker for calcium chloride (CaCl2) precipitation. Under constant stirring, CaCl2 was added dropwise. The final concentration of the CaCl2 was 100 mM. To enrich the crude membrane proteins, 2 mL of the homogenate was centrifuged at 25,830 g for 60 min at 4 °C. To separate the basolateral membranes from the apical membrane proteins, centrifugation of 7.5 mL of the homogenate at 2,000 g for 30 min at 4 °C was performed. A total of 4 mL of the outcoming supernatant was again centrifuged at 25,830 g for 60 min at 4 °C to enrich apical membrane proteins. Resuspension buffer (10 mM TRIS base, 150 mM NaCl, pH 7.4) was admixed with inhibitors of phosphatases (1:100) and proteases (1:50) (Protease Inhibitor Cocktail [P8340], Phosphatase Inhibitor Cocktail [P5726], Sigma-Aldrich Chemie GmbH, Munich, Germany). Subsequently, supernatant was removed after centrifugation and resuspension buffer added to the pellet. After resuspending the pellets, tissue homogenate was aliquoted and stored at -20 °C until use.
For preparing the cytosol needed for Hif-1α detection, 50 mL cytosol-buffer (150 mM NaCl, 1%
Nonidet™P 40 [Sigma-Aldrich Chemie GmbH, Munich, Germany], 50 mM TRIS base, 5 mM EDTA) was admixed with 250 µL phenylmethansulfonyl fluoride (PMSF, Sigma-Aldrich Chemie GmbH, Munich, Germany). An amount of 0.2 g of tissue was incubated on ice with 1.85 mL of buffer until thawed.
Afterwards, tissue samples were homogenised with the Potter grinder and then centrifugated at 4 °C for 30 min at 17,500 g, followed by aliquoting of the supernatant and storage at -20 °C.
The Bradford method in duplicates with the Bradford reagent (SERVA Electrophoresis GmbH, Heidelberg, Germany) was used to measure the protein content.
2.6.2. Western Blot – Performance
Tissue samples were resolved under reducing conditions in Tris-glycine buffer (125 mM TRIS HCl, 3%
SDS, 10% glycerol, 0.1 M DTT, 5% bromphenol blue) for denaturation. Conditions for the denaturation as well as the used preparation methods can be seen in Table 4.
Table 4. Western Blot conditions and used antibodies
After denaturation, the protein solution was centrifugated to detach condensed water from its lid and 10 µL of each solution was applied on an 8.5 % SDS-polyacrylamide gel. For identifying the protein sizes, 26616 PageRuler™ (ThermoFisher Scientific Inc, Waltham, MA, USA) was used. Electrophoresis was performed for 60 minutes with the help of Tris-glycine buffer (250 mM TRIS base, 1.92 M glycine) containing 1% sodium dodecyl sulphate (SDS). Subsequently, proteins were transferred onto nitrocellulose membranes (Amersham Biosciences Europe GmbH, Freiburg, Germany) via electroblotting technique (tank blot procedure).
Antibody Prep Denaturation Protein
loading Blocking PAK (O/N
*diluted in BSA (5%) instead of 5 % Milk/TBST; **diluted in 2.5 % Milk/TBST; ***diluted in 2.5 % Milk/PBST AM = apical membrane; CM = crude membrane; CY = cytosol
Blotted membranes were blocked in 5% milk (5 g milk powder in tris-buffered saline supplemented with 1% tween [TBST]) for 60 min at room temperature. Membranes used for Hif-1α detection were blocked with 5% milk dissolved in PBST (phosphate-buffered saline supplemented with 1% Tween 20).
Membranes were incubated overnight with the respective primary antibody (Tab. 4) on a shaker at 4 °C .
Membranes were washed (3 x 10 min, TBST/ PBST for Hif-1α) and secondary antibodies, which were labelled with horseradish-peroxidase, were applied for 90 min. During this time, immunocomplexes of primary and secondary antibodies were formed on the membrane. After washing the membranes again (3 x 10 min, TBST/ PBST for Hif-1α), enzymes, which were linked to the immunocomplexes, were detected by using SuperSignal® West Dura Extended Duration Substrate for ASCT1, PepT1, SGLT1 and Na+/K+-ATPase (ThermoFisher Scientific, Waltham, USA). For the detection of pSGLT1, GLUT2 and Hif-1α, SuperSignal® West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific Inc., Waltham, USA) was used. Signals of both substrates were registered by the ChemiDoc™MP imaging system and analysed with Image Lab 5.2.1. software (both Bio-Rad Laboratories GmbH, Feldkirchen, Germany).
To detect the phosphorylated SGLT1, membranes incubated with the pSGLT1 antibody were stripped (200 mM glycine, 0.05% Tween 20 and 1% SDS for 45 min) in between the first antibody detection and housekeeper protein detection, followed by exposure to the SGLT1-specific antibody. This way, a more precise comparison between SGLT1 and its phosphorylated form was enabled. Performance of the application of antibodies and substrate detection was the same as previously described. Used antibodies can be found in Table 4.
Indian ink (royal blue ink combined with 2% of glacial acetic acid) staining for 30 min and subsequent destaining with distilled water were performed to control protein loading of the applied tissue homogenates. Indian ink was used as loading control.
2.6.3. Western Blot – Analysis
To determine the band intensities, densiometric measurement was performed via Image Lab 5.2.1.
software (Bio-Rad Laboratories GmbH, Feldkirchen, Germany).
Molecular masses were determined as follows: SGLT1 showed a mass of ~ 90 kDa, its phosphorylated form pSGLT1 a mass of ~ 95 kDa, GLUT1 a mass of ~ 58 kDa, Na+/K+-ATPase a mass of ~ 110 kDa, PepT1 a mass of ~ 79 kDa and Hif-1α a mass of ~ 130 kDa.
For the amino acid transporter 1, two possible molecular masses were visible during the laboratory work: after adding the used antibody, a band appeared at 65 kDa as well as at 40 kDa. At the end, the
65 kDa band was taken for evaluation based on the literature (Talukder et al. 2008) and in accordance with the manufacturer’s recommendations.
Figure 3. Representative Western Blot taken from individual pigs. Expected molecular masses are indicated with an arrow.
The first six bands always represent the control animals. The following six bands represent the infected animals.