Analysis of proteins by SDS-PAGE and Western Blotting

2.8.1 Preparation of crude extract of macroplasmodium

1) After the macroplasmodium was harvested, weighed and transferred into a glass homogenizer, 2 volumes of lysis buffer, 1/25 volume (as calculated from volume of lysis buffer) of protease inhibitor cocktail and 1/1000

volume (as calculated from total volume) of Mercaptoethanol were added.

2) After the cells were well homogenized, the sample was transferred into a clean tube and was centrifuged at 17000 rpm for 30 min to clarify the lysate.

3) The supernatant was carefully removed without disturbing the pellet and was centrifuged again for 15 min.

4) The lysate was either immediately used for analysis or was frozen in liquid nitrogen and stored at -80°C for later use.

2.8.2 Determination of protein concentration using the Bradford assay

1) 2, 5, 7, 10, 15, 20 and 50 µl of BSA stock solution (0.1 mg/ml) was added to the cuvettes used for the standard curve.

2) An appropiate volume of millipore H2O was added to each cuvette from step1, to give total volume of 0.1 ml.

3) A 1:5 dilution from the Bradford stock solution was performed with millipore H2O 0.9 ml of the diluted Bradford stock solution was added to every cuvette (from step 1), producing a total volume of 1 ml.

4) 1 µl of crude extract was added to a cuvette and made up to 0.1 ml with millipore H2O. 0.9 ml of the diluted Bradford reagent solution was then added.

5) All the samples were incubated at room temperature for 20 min. Protein measurement was then carried out at a wavelength of 595 nm in a UV-Vis spectrophotometer.

2.8.3 SDS-PAGE

1) The glass plates and spacers were thoroughly cleaned with detergent and ddH2O. The air dried glass plates and spacer were wiped using paper prewetted with 100% ethanol.

2) The spacers were transferred between the two glass plates and fix with clamps, forming a sandwich.

3) The seperation gel mixture was prepared with ammoniumsulfate and TEMED adding at the end. The mixture was briefly vortex and immediately filled into the sandwich up to about 4 cm from the top edge.

4) About 0.5 ml of isopropanol was immediately loaded to make the surface of the acrylamid gel mixture even. The gel was allowed to polymerize for about 20 min. A sharp gel-alcohol interface should be seen once the gel has polymerized.

5) The isopropanol layer was then drained away by inverting the casting unit.

6) The stacking gel was then prepared, mixed and added into the sanwich.

The comb was then carefully inserted into the sanwich.

7) While the stacking gel was polymerizing, the protein samples and the

volume of 2x sample loading buffer was added to eaach sample and standard marker. The samples were then incubated at 95°C for 10 min.

8) After the stacking gel was polymerized the comb was carefully pulled from the gel and the sandwich was tightly casted into the electrophorese apparatus and the chambers were filled with 1x running buffer.

9) The wells and the bottom of the sandwich were rinse well with the running buffer to remove any air bubbles.

10) The samples together with the standard marker were then loaded. The apparatus was connected to a power supply. First 10 mA was applied for one gel (1.5 mm-thick standard sized gel) until a sharp line was observed.

The power was then turned on to 25 mA.

11) The gel was allowed to run until the dye reached about 1 cm from the bottom of the gel.

2.8.4 Western Blotting

1) A piece of polyvinylidene difluoride (PVDF) and 6 sheets of Whatman filter paper to the same size as the gel were cutted. The Whatman filter papers were soaked in transfer buffer for 5 min prior to blotting. The PVDF membrane was soaked in methanol for 15 s and later in distilled water for 2 min, prior to soaking in transfer buffer.

2) A blotting system was assemblied in the order shown in Figure 16:

3 sheets of the soaked Whatman papers were transferred to the plate. Any bubles were removed by rolling a pipette over the papers. The PVDF membran was then placed on the top of the flter papers the membran was carefully overlaid with the gel. The remaining 3 sheets of filter papers were placed on the gel. The top plate was placed on top of the assembled unit.

3) A bottle containing 1 liter liquid was placed on the top of the plate to serve as a weight.

4) The assembled unit was connected to a power supply. Blotting was allowed to proceed for 1h at 2 mA/cm² of the membrane.

cathode

Figure 16 Diagram of semidry electroblotting

5) The membrane was then strained with Ponceau solution for 2-3 min. The membrane was washed with a small amount of distilled water and shaken untill the standard marker was visible.

The membrane was washed two times with 1xPBS/Tween buffer and one time with 1x PBS buffer for 10 min each wash..

7) 20 ml of blocking buffer was added and the membrane was incubated overnight at 4°C.

8) The membrane was washed as in step 6 and incubated with the primary antibody (diluted in blocking buffer) for 2 h at room temperature with shaking.

9) After washing as in step 6, the membrane was incubated with diluted secondary antibody (conjugated with horseradish peroxidase) for 1 h at room temperature with shaking.

10) The mambrane was then washed four times with PBS/Tween buffer and one time with PBS buffer for 10 min each wash.

11) The proteins were detected by a chemiluminescent reaction of peroxidase-conjugated antibody. For this purpose, the membrane was placed on a tissue paper. 1.5 ml of each Nowa A and Nowa B solution was mixed. The membran was overlaid with the mixture. After incubation at room temperature for 1 min, the membrane was overlaid with a plastic foil and was transferred into an exposure cassette.

12) In a darkroom, an chemoluminescent detection film was placed on the plastic foil and the cassette was closed immediately. The exposure was allowed to proceed first for 5 s, to reduce any background.

13) The film was placed into an automatic developer. Step 12 was repeated again with increasing the exposure time to 10 s.