Horseradish peroxidase hrp

Total protein was isolated from cultured cells using protein lysis buffer (Thermo). Protein concentration was determined by BCA assay (Thermo). Equal amounts of protein (100 ug) from cell extracts were subjected to 4% to 15% gradient SDS-PAGE gels (Bio-Rad) and subsequently transferred to nitrocellulose membranes. After blocking for 1 h with TBS containing 0.1% Tween20 and 5% non-fat dry milk, membranes were incubated with primary antibodies. Secondary antibody conjugated with horseradish peroxidase (HRP) (GE Healthcare) was used at 1:2000 dilution to detect primary antibodies, and enzymatic signals were visualized by an enhanced chemiluminescence system (Amersham Biosciences).

Dissected cerebellum was lysed with SDS lysis buffer (1% SDS, 10% glycerol, 5% β-mercaptoethanol and 125 mM Tris-HCl, pH 6.8). The tissue was homogenized and then boiled at 100 °C for 10 min. To extract proteins from HEK293A cells, the cells were lysed with NP-40 lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl and 50 mM Tris-HCl, pH 7.5, with Complete protease inhibitor cocktails (Roche)). All lysates were centrifuged and the supernatant were collected for separation by 10% SDS–polyacrylamide gel electrophoresis. The separated proteins were blotted to polyvinylidene difluoride membrane by Bio-Rad Trans-Blot Turbo Transfer System. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature. The blocked membrane was incubated with 1:1,000 mouse anti-Espin (BD Biosciences, 611656) or 1:1,000 rabbit anti-β-actin (Cell Signaling, 4967S) or 1:1,000 rabbit anti-FLAG (Sigma, F7425) diluted in 2% ECL Prime Blocking Reagent (GE Healthcare) at 4 °C overnight. After washing, the membrane was incubated with respective secondary antibodies conjugated to horseradish peroxidase (HRP) (GE Healthcare) for 1 h at room temperature. The signal was detected using ECL Western Detection Reagent (GE Healthcare) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).

Proteins were separated on acrylamide gels as described above and transferred to methanol-activated polyvinylidene difluoride (PVDF) membranes (Merck, IPVH00010), using Western blot transfer buffer (3.03 g Tris, 14.4 g glycine, 200 ml methanol [all per 1 liter]) for 1.5 h at 100 V. All the incubation steps from here on were performed with constant moderate agitation by using rocking platforms. Membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) (20 mM Tris, 10 mM NaCl, 0.1% Tween 20) and then incubated with appropriately diluted primary antibodies (anti-BamA diluted 1:10,000 and anti-SecG diluted 1:6,000) in 5% skim milk in TBST overnight at 4°C. After three 5-min washes with TBST, membranes were incubated for 1 h with secondary anti-rabbit antibodies conjugated with horseradish peroxidase (HRP) (catalog no. NA934; GE Health Care) diluted 1:10,000 in 5% skim milk in TBST. After these antibody incubations, membranes were washed again three times for 5 min with TBST. Proteins were detected by adding ECL substrate (catalog no. RPN2106; GE Healthcare), then exposing and visualizing using a digital developing machine (ChemiDoc touch imaging system).

Cerebella were dissected from mice and lysed in RIPA lysis buffer [50mM Tris HCl, pH7.4, 150mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.2% SDS, phosphatase (Sigma) and protease inhibitors cocktail (Roche)]. After three cycles of freeze and thaw, proteins were separated on a 12% or 15% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The following primary antibodies were used: anti-ATXN1 (rabbit 11NQ, Orr lab), anti-PSD95 (Biolegend), and alpha-tubulin (mouse, Sigma). Signals from secondary antibodies linked to horseradish peroxidase (HRP) (GE Healthcare) were detected using Amersham ECL Western Blotting Detection Reagent (GE Healthcare) and ImageQuant LAS 4000 imager (GE Healthcare); protein levels were quantified using ImageQuant (GE healthcare) and ImageJ. Data was analyzed with one-way ANOVA followed by Bonferroni post-hoc test.

Total cell lysates were prepared and equalized for total protein concentration, as previously described (51 (link)). Briefly, protein homogenates were mixed with sample buffer [160 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM DTT, and 0.005% Bromphenol Blue], boiled, and subjected to SDS-PAGE and electrotransferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were blocked with PBS-T (PBS, 05% Tween 20) and 5% nonfat dry milk for 1 h at room temperature, followed by individual incubation with primary antibodies in PBS + 1% BSA overnight at 4 °C under gentle agitation. After that, the membranes were washed 5 times with PBS-T and incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) (GE Healthcare) in PBS-T (PBS, 05% Tween 20) and 5% nonfat dry milk for 1 h at room temperature. The membranes were washed five times with PBS-T and proteins were detected using enhanced chemiluminescence solutions [solution 1: 1 M Tris–HCl (pH 8.5), 250 mM luminol, 90 mM p-coumaric acid; and solution 2: 30% H₂O₂, 1 M Tris-HCl (pH 8.5)] and visualized with the ChemiDoc imaging system (GE Life Science).