Transcription Factor Sequestration by Polyglutamine Proteins - Protein Misfolding and Cellular Stress in Disease and Aging - page 209

6. pcDNA3.1 expression vector with transcriptional factor
cDNA tagged with V5.

1. Phosphate buffered saline (PBS).
2. 2× SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% sodium
dodecyl sulfate (SDS), 20% glycerol, 0.01% bromphenol blue,
10% beta-mercaptoethanol).
3. Microsonicator.
4. Materials for SDS-PAGE and Western blotting (polyacrylam-
ide gel, polyvinylidene difluoride (PVDF) membrane, run-
ning buffer, blotting buffer).
5. TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05%
Tween20).
6. Dried skim milk.
7. Goat serum.
8. Mouse monoclonal antibodies against GFP (04363-66; naka-
lai tesque) and V5 tag (R960-25; Invitrogen).
9. Horseradish peroxidase (HRP) conjugated anti-mouse IgG.
10. Chemiluminescent reagent.
11. X-ray film or video camera system.

2.1.2. Materials for
Western Blot Analysis

1. Phosphate buffered saline.
2. 2× SDS sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS,
20%

2.1.3. Materials for Filter
Trap Assay

glycerol, 0.01% bromphenol blue, 10% beta-

mercaptoethanol).
3. Cellulose acetate membrane with 0.2 mm pore size.
4. Dot blotting apparatus.
5. Wash buffer (2% SDS, 50 mM Tris-HCl, pH 8.0, 10% Glycerol).
6. TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05%
Tween20).
7. Dried skim milk.
8. Goat serum.
9. Mouse monoclonal antibodies against GFP (04363-66; naka-
lai tesque) and V5 tag (R960-25; Invitrogen).
10. HRP conjugated anti-mouse IgG.
11. Chemiluminescent reagent.
12. X-ray film or video camera system.

1. 4 well chamber slide.
2. Phosphate buffered saline.
3. 4% paraformaldehyde (PFA) in PBS.

2.1.4. Materials for
Immunofluorescence
Microscopy