4.3.1 Preparation of cell lysates for immunoblot analysis
For the preparation of lysates for immunoblots cells were harvested by detaching them without trypsin or if cell surface molecules on adherent cells were analysed, the cells were scratched off. Cells were counted using a Neubauer counting chamber and washed 4 times
4 Methods
with PBS. After the last washing step, the remaining liquid was removed by pipetting and cell pellets were frozen at -20◦C. 15µl reducing sample buffer (see table 3.7 on page 28) were added per 1×105 cells, the samples were boiled for 5 min at 95 ◦C in a water bath, shortly vortexed afterwards and centrifuged for 5 min at 10 000 x g to get rid of cell debris.
Usually 40 µl of each sample were loaded onto a SDS gel. For the lysates for the CAR analysis, it was essential that the cells were seeded in the same densities as the expression of HSPGs were described to vary with cell density (Dechecchi et al. 2001).
4.3.2 SDS-PAGE
One way of separating proteins is according to their size and charge. In the polyacryl-amide gel electrophoresis (PAGE) (Laemmli 1970) in the presence of SDS secondary and non-disulfide-linked tertiary structures of the native protein are destroyed. The an-ionic detergent SDS adds negative charge to the protein. To reduce disulfide bridges β-mercaptoethanol is added. SDS binds uniformly to all proteins conferring a mass:charge ratio, which is proportional to the size of the protein. Upon application of an electric field proteins are then separated according to their size. Polyacrylamide is used, as it is thermo-stable, chemically relatively inert, easy to polymerise, and the pore size can be modified by the amount of TEMED added.
All buffers and solutions necessary for SDS-PAGE and immunoblot analysis are listed in table 3.7 on page 28. The separating gel was poured into a chamber consisting of two glass plates, which were separated by spacers and fixed with clamps. After polymerisation a stacking gel was poured on top, into which a comb was inserted for forming of pockets.
After polymerisation of the stacking gel, the gel was fixed in a vertical electrophoresis chamber, both buffer reservoirs were filled with running buffer and air bubbles were re-moved. The lysates were loaded onto the gel with a Hamilton syringe. The electrophoretic separation was performed at 40 mA until the dye front reached the bottom of the gel.
4.3.3 Immunoblot
Immunoblot is a technique to transfer proteins after a SDS-PAGE from the polyacrylamide gel onto a nitrocellulose membrane by applying an electric field (Towbin et al. 1979). The proteins are fixed on the membrane like a copy of the gel and are freely accessible by antibodies.
After the SDS-PAGE, the gel was placed into transfer buffer for equilibriation. The blot was set up in the following order, whereby except for the outermost perforated plastic plates, everything else was soaked in transfer buffer: perforated plate, sponge, blotting paper, gel, nitrocellulose membrane, blotting paper, sponge, perforated plate. The stack was placed into a vertical chamber at 4 ◦C containing transfer buffer. Perpendicular to
4 Methods
the gel an electric field was applied so that the negatively charged proteins in the gel were transferred onto the nitrocellulose membrane. It was therefore important, that no air bubbles are between the gel and the nitrocellulose membrane, when the stack is set up. Transfer was done overnight at 16 V.
After transfer, the gel was removed from the stack and equilibriated in PBS/Tween.
To determine whether the blotting procedure was successful, the blot was stained with Ponceau S, an azo dye, which binds reversibly to the positively charged amino groups of proteins. Protein lanes and bands of a length standard were marked with a ball-pen and the blot was cut to a proper size. Depending on the antibodies used for develop-ing the immunoblot, the blot was cut into an upper and a lower part for development with two different antibodies, or left as one blot for development with just one antibody.
The addition of antibodies to the blot was always followed by washing off unbound an-tibodies with PBS/Tween. The primary antibody was either added for 2 hrs at RT in PBS/Tween or overnight at 4 ◦C. Afterwards, blots were washed 3 times for 5 to 10 min with PBS/Tween on a shaker before the secondary antibody was added for 60 min at RT.
This second incubation time was followed by 3 washing steps for at least 10 min each with PBS/Tween.
The blots were either developed with DAB or with the more sensitive method ECL. The addition of reduced DAB and H2O2 for developing the immunoblot leads to an oxidation of DAB and the formation of water by the enzyme peroxidase (HRP), which is conjugated to the secondary antibody (see equation 4.4). The oxidation of DAB results in a brown colour at the site where the peroxidase converts the DAB, which is then visible on the blot.
DABred+ H2O2+ 2H+
peroxidase
GGGGGGGGGGGGGGGADABox+ 2H2O (4.4) The development of the immunoblot with ECL was done by addition of luminol and H2O2, which are oxidised by the HRP to an excited form of 3-aminophthalate (3-APA) (see equation 4.5). The decay of the excited form of 3-APA to a lower energy state is responsible for the emission of light. With this method proteins can be visualised even, if they are present in femtomole quantities and the light signal can be captured by exposing the blot to the camera of an imaging system (Chemilux system from Intas).
luminol + H2O2
peroxidase
GGGGGGGGGGGGGGGA3−APAexcited
peroxidase
GGGGGGGGGGGGGGGA3−APA + light (4.5) Primary antibodies were diluted as follows: 1:500 for CAR, 1:4000 for HSC70, 1:1000 for caspase-8. The secondary antibodies were conjugated with HRP and were diluted 1:10000 for ECL development and 1:4000 for DAB development.
4 Methods
4.3.4 Densitometric analysis of levels of coxsackie and adenovirus receptor on Sulf mouse embryonic fibroblasts
A densitometric analysis was performed, to compare levels of CAR with the internal loading control HSC70. This analysis allows to compare defined band intensities/volumes and depicts them as an integral of optical density. The internal control, in our case HSC70, was set to 100 % so that the ratios of the integrals of optical density for CAR could be calculated. The programm used was gel pro analyzer 4.5 from Media Cybernetics.