- Protein concentration is measured in supernatants by Lowry
method using BIO-RAD protein assay kit in a 96-well plate. - 20μg of total protein is diluted with H 2 Oto20μl total
volume, mixed with 5μlof4loading buffer and heated at
100 C for 3 min. Samples are ready for loading on SDS PAGE
(seeNote 22).
SDS polyacrylamide gel electrophoresis
- For a 10% SDS PAGE, mix 2.65 ml of H 2 O, 3.35 ml of
acrylamide/bisacrylamide solution, 500μl of SDS (5%), and
3.75 ml of Tris-HCl (1 M, pH 8.8). Polymerization is started
by addition of 75μl of 10% (w/v) APS and 15μl of TEMED. - For the 3.6% stacking gel, mix 1.75 ml of H 2 O, 0.5 ml of
acrylamide/bisacrylamide solution, 100μl of 5% SDS, and
2.5 ml of Tris–HCl (0.25 M, pH 6.8). Polymerization is started
by addition of 75μl of 10% (w/v) APS and 10μl of TEMED. - The samples are loaded and separated electrophoretically at
150 V until the protein reach the running gel and then at
200 V for about 1 h until the bromophenol dye front run off
the gel.
Immunoblotting
- After electrophoresis, separate the gel plates and remove the
stacking gel. - The PVDF membrane is immerged in ethanol and then in H 2 O
to make it hydrophilic. - The 10% gel can be then blotted according to the wet blotting
method. - First, two sponges, six Whatman filter papers, and the PVDF
membrane are equilibrated in blotting transfer buffer for
30 min. - The holder cassette is set inside the container containing blot-
ting transfer buffer with the black side facing down. - Then the apparatus is prepared as followed from the cathode:
one sponge, three filter papers, the gel, the PVDF membrane,
three filter papers, and one sponge. - Bubbles are removed by rolling over filter paper using a 15 ml
tube. The holder cassette is then closed and placed in the
container (seeNote 23). - The container is placed into the blotting chamber. Place a
cooling unit into the chamber, fill with blotting transfer buffer. - The blotting is performed for 1 h at 100 V.
Antibodies - After the transfer, incubate the PVDF membrane with block-
ing solution for 1 h at room temperature on a stirring plate.
Study of Cardiac Hypertrophy and Protein Synthesis 333