Iblot gel transfer stacks pvdf

Cells were lysed and total protein extracted using the Minute™ Total
Protein Extraction Kit for Animal Cultured Cells and Tissues (Invent
Biotechnologies, Inc.) according to the manufacturer's protocol. The
Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to determine
protein concentration.
Proteins were separated on NuPAGE™ 3%–8% Tris-Acetate
Gels (Invitrogen) (for detection of HTT and GAPDH) or NuPAGE
4%–12% Bis-Tris Gel (Invitrogen) (for detection of cleaved
caspase 3) at 150V for 1 h and electrotransferred onto polyvinylidene
difluoride (PVDF) membranes (iBlot^® Gel Transfer Stacks PVDF,
Invitrogen) using iBlot system (Invitrogen). The membranes were blocked using
5% nonfat dry milk diluted in 0.1% Tween 20/PBS. Primary and
secondary Abs (Supplementary
Table S3) diluted in blocking solution were added for incubation
overnight at 4°C and 1 h RT, respectively. Primary Ab directed
against HTT detects both mHTT (upper band) and wtHTT (lower band) protein. The
bands were detected using SuperSignal™ West PicoPLUS Chemiluminescent
Substrate (Thermo Scientific) and visualized using ImageQuant™ LAS4000
(GE Health care). Each technical treatment replicate was blotted three times and
differences in HTT and GAPDH protein levels were quantified using ImageJ
software.

Total protein extracts from PC3-DR cells were separated by SDS-PAGE (NuPAGE 4–12%, ThermoFisher Scientific) followed by transfer to iBlot Gel Transfer Stacks PVDF (Invitrogen Cat# IB24002) in the Invitrogen iBlot 2 transfer system. Membranes were blocked overnight with 5% dry milk in TBS-T buffer (20 mM Tris–HCl, pH 7.6, 140 mM NaCl, 0.2% Tween 20) and then probed with individual human sera or commercial antibodies for 2 h. Membranes were washed at least three times with TBS-T and then incubated for 2 h with the respective horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-human, Invitrogen, Cat# A18847; goat anti-rabbit, Cell Signaling Technology, Cat# 7074S). After several washes with TBS-T, detection of antibodies bound to proteins was achieved by enhanced chemiluminescence (Fisher Scientific, Cat# PI34580). Protein bands were captured in X-ray films, and images of Western blots were generated by scanning the films in an Epson WF2830 scanner.