CHAPTER II MATERIALS AND METHODS 2.1 Chemicals and ...

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through polyacrylamide gel. Smaller proteins migrate faster through this mesh and
the proteins are thus separated according to size.
The cells were treated in the same manner as in the RT-PCR analysis. After
treatment, the cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 1
mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, and 5 mM mercaptoethanol).
The protein (30 µg) from the cell lysates was separated by 8% SDS-polyacrylamide
gel electrophoresis and was transferred onto a nitrocellulose membrane by
electroblotting. The membrane was probed with the indicated primary antibody.
Following incubation with enzyme-linked secondary antibody, signal was detected by
enhanced chemiluminescence (Thermo Scientific) and captured on Kodak X-ray film
(Figure 2.4).
Figure 2.4 The schematic of SDS-PAGE and Western blot.