3. Protease inhibitor cocktail (1Â PIC): 1 tablet/100 mL buffer
(SigmaFAST EDTA free).
4. Blocking buffer: 1Â Roti
®
-block buffer (Carl Roth).
2.2 Radiobinding
Assay
1. MicroBeta filtermat 96-cell harvester (Perkin-Elmer).
2. MicroBeta
2 microplate counter (Perkin-Elmer).
3. PEI-coated GF/B glass fiber filter-bottom 96-well microplate
with BackSeal and TopSeal (Perkin-Elmer).
4. Ultima Gold MV liquid scintillation cocktail (Perkin-Elmer).
5. Wash buffer: 50 mM Tris–HCl, pH 7.4.
6. Radioligand solution: 3.6 μM
3 H-CGS21680 (Perkin-Elmer)
in wash buffer.
7. Nonspecific ligand solution: 3.6 μM
3
H-CGS21680, 1 mM
CGS21680 in wash buffer.
8. γ-Globulin solution: 0.1% (w/v) γ-globulin in wash buffer.
9. PEG6000 solution: 25% (w/v) PEG6000 in wash buffer.
Fig. 3 Membrane protein behavior in solution. (a) Native-PAGE on purified Matrix 2 ion channel revealed by
western blot. (b) Chemical cross-linking assay using different amounts of glutaraldehyde, analyzed by
SDS-PAGE and revealed by Western blot. (c) Gel filtration (5-150GL) profile and corresponding Western blot
analysis. (Adapted from [13]). A similar approach was also previously reported for the characterization of the
human transcription factor TFIIE [14]
260
Vincent Corvest and Anass Jawhari
(SigmaFAST EDTA free).
4. Blocking buffer: 1Â Roti
®
-block buffer (Carl Roth).
2.2 Radiobinding
Assay
1. MicroBeta filtermat 96-cell harvester (Perkin-Elmer).
2. MicroBeta
2 microplate counter (Perkin-Elmer).
3. PEI-coated GF/B glass fiber filter-bottom 96-well microplate
with BackSeal and TopSeal (Perkin-Elmer).
4. Ultima Gold MV liquid scintillation cocktail (Perkin-Elmer).
5. Wash buffer: 50 mM Tris–HCl, pH 7.4.
6. Radioligand solution: 3.6 μM
3 H-CGS21680 (Perkin-Elmer)
in wash buffer.
7. Nonspecific ligand solution: 3.6 μM
3
H-CGS21680, 1 mM
CGS21680 in wash buffer.
8. γ-Globulin solution: 0.1% (w/v) γ-globulin in wash buffer.
9. PEG6000 solution: 25% (w/v) PEG6000 in wash buffer.
Fig. 3 Membrane protein behavior in solution. (a) Native-PAGE on purified Matrix 2 ion channel revealed by
western blot. (b) Chemical cross-linking assay using different amounts of glutaraldehyde, analyzed by
SDS-PAGE and revealed by Western blot. (c) Gel filtration (5-150GL) profile and corresponding Western blot
analysis. (Adapted from [13]). A similar approach was also previously reported for the characterization of the
human transcription factor TFIIE [14]
260
Vincent Corvest and Anass Jawhari