2.4.2 Western Blot
Analysis
1. 150-mm Petri dishes (Falcon
® 150 mm TC-treated dishes,
Corning, cat. no. 353025).
2. Cell scrapers (Dominique Dutscher, cat. no. 353085).
3. PBS containing 30% w/v glycerol (Sigma Aldrich, cat.
no. G5516).
4. RIPA buffer: 20 mM Tris-HCl or HEPES, pH 7.5, 120 mM
KCl, 1% NP-40, 0.1% SDS, 1 mM EDTA, 0.5%
Na-deoxycholate, supplemented with protein inhibitor cocktail
(Roche™) and 0.5 mM 1,4-dithreothiol (DTT) (Sigma
Aldrich).
5. Lysis buffer: 20 mM Tris or HEPES, 250 mM KCl, NP-40
0.05% supplemented with protein inhibitor cocktail (Roche™)
and 0.5 mM DDT.
6. Protein assay dye reagent (Bio-Rad, cat. no. 5000006).
7. Laemmli buffer 4Â: 60 mM Tris-HCl, pH 6.8, 10% glycerol,
2% SDS, 0.0005% Bromophenol Blue, 355 mM
β-mercaptoethanol.
8. SDS PAGE gels, PVDF transfer membranes, 3MM Whatman
paper.
9. TBST: 20 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% Tween20.
10. Non-fat dry milk or BSA powder.
11. Western blotting detection reagents (Amersham ECL Prime,
cat no. 3030-931).
12. Primary antibodies against the target protein or against the
GFP tag and corresponding secondary antibodies coupled to
horseradish peroxidase (HRP). For the detection of XPB protein, we used a mouse monoclonal anti-XPB antibody (1B3,
MABE1123, Sigma-Aldrich) and a donkey anti-mouse antibody coupled with HRP.
2.4.3 Fluorescence
Microscopy
1. Glass-bottom dishes (Glass Bottom Dish 35 mm, Clinisciences/Ibidi, cat. no. 81218-200).
2. Epifluorescence inverted microscope with filters compatible
with DAPI/Hoechst and the fluorescent reporter used (here
GFP).
3. Paraformaldehyde (PFA) 16% ultrapure methanol free (Euromedex, cat. no. 15710).
4. Hoechst (Sigma Aldrich, cat. no. 32670).
5. Antibodies to confirm the expression of the target protein or
for co-localization experiments.
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
43
Analysis
1. 150-mm Petri dishes (Falcon
® 150 mm TC-treated dishes,
Corning, cat. no. 353025).
2. Cell scrapers (Dominique Dutscher, cat. no. 353085).
3. PBS containing 30% w/v glycerol (Sigma Aldrich, cat.
no. G5516).
4. RIPA buffer: 20 mM Tris-HCl or HEPES, pH 7.5, 120 mM
KCl, 1% NP-40, 0.1% SDS, 1 mM EDTA, 0.5%
Na-deoxycholate, supplemented with protein inhibitor cocktail
(Roche™) and 0.5 mM 1,4-dithreothiol (DTT) (Sigma
Aldrich).
5. Lysis buffer: 20 mM Tris or HEPES, 250 mM KCl, NP-40
0.05% supplemented with protein inhibitor cocktail (Roche™)
and 0.5 mM DDT.
6. Protein assay dye reagent (Bio-Rad, cat. no. 5000006).
7. Laemmli buffer 4Â: 60 mM Tris-HCl, pH 6.8, 10% glycerol,
2% SDS, 0.0005% Bromophenol Blue, 355 mM
β-mercaptoethanol.
8. SDS PAGE gels, PVDF transfer membranes, 3MM Whatman
paper.
9. TBST: 20 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% Tween20.
10. Non-fat dry milk or BSA powder.
11. Western blotting detection reagents (Amersham ECL Prime,
cat no. 3030-931).
12. Primary antibodies against the target protein or against the
GFP tag and corresponding secondary antibodies coupled to
horseradish peroxidase (HRP). For the detection of XPB protein, we used a mouse monoclonal anti-XPB antibody (1B3,
MABE1123, Sigma-Aldrich) and a donkey anti-mouse antibody coupled with HRP.
2.4.3 Fluorescence
Microscopy
1. Glass-bottom dishes (Glass Bottom Dish 35 mm, Clinisciences/Ibidi, cat. no. 81218-200).
2. Epifluorescence inverted microscope with filters compatible
with DAPI/Hoechst and the fluorescent reporter used (here
GFP).
3. Paraformaldehyde (PFA) 16% ultrapure methanol free (Euromedex, cat. no. 15710).
4. Hoechst (Sigma Aldrich, cat. no. 32670).
5. Antibodies to confirm the expression of the target protein or
for co-localization experiments.
Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System. . .
43