Transcription Factor Sequestration by Polyglutamine Proteins - Protein Misfolding and Cellular Stress in Disease and Aging - page 213

Fig. 1. Example data of Western blot analysis showing interaction of transcriptional factor
with SDS-insoluble aggregates in Neuro2a cells. Neuro2a cells were transfected with
expression vector for NF-YA or LacZ (control) tagged with V5 together with expression vector
for Nhtt18Q-EGFP-NLS or Nhtt150Q-EGFP-NLS. After 24 h, cells were lyzed and subjected
to SDS-PAGE and Western blot analysis using anti-GFP or anti-V5 antibody. Bands for
expressed proteins are indicated by arrows and positions of the gel top are indicated by
arrowheads . Note that gel top bands were detected by anti-GFP and anti-V5 staining in the
cells expressing Nhtt150Q-EGFP-NLS but not ones expressing Nhtt18Q-EGFP-NLS.
more sensitive and quantitative than Western blot analysis
described above to analyze the aggregate proteins in the cell lysates.
1. Wash the transfected cells with PBS and harvest the cells with
150 ml of 2× SDS sample buffer.
2. Heat the cell lysates at 100°C for 5 min.
3. Dilute 10 ml of cell lysates with 40 ml of 2× SDS sample
buffer.
4. Pre-wet a cellulose acetate membrane and two filter papers
with wash buffer.
5. Put the membrane on two filter papers and set them into the
dot blotting apparatus.
6. Apply 50 ml of diluted lysates to each well and start suction
(see Note 5).
7. Apply 300 ml of wash buffer twice to wash out soluble materi-
als from the membrane.
8. Keep the suction for 20 min (see Note 6).