Amersham ecl western blotting detection kit

Whole spinal cord protein samples from α₂^+/G301R and α₂^+/+ littermate mice exposed to SCI and allowed 3 days survival in addition to naïve α₂^+/G301R and α₂^+/+ littermate mice were prepared as described [50 (link)]. Equal amounts of protein were separated by SDS-PAGE on 10–14% (α_1, α₂, α₃ and AQP4) gels and electro-blotted onto nitrocellulose membranes (Pharmacia-Amersham). Membranes were blocked in PBS with 5% skimmed milk and 0.5% Tween-20 and incubated with the following primary antibodies: anti-α₁ diluted 1:2000 (clone a6f-c, Developmenal Studies Hybridoma Bank), anti-α₂ diluted 1:1000 (Merck Millipore), anti-α₃ diluted 1:1000 (Merck Millipore), anti-AQP4 diluted 1:1000 (AQP-004, Alomone labs) anti-GAPDH diluted 1:1000 (Abcam), or anti-β-Actin diluted 1:2000 (Sigma-Aldrich) overnight at 4 °C.
Next, membranes were incubated with HRP-conjugated secondary antibodies (swine anti-rabbit HRP diluted 1:2000 (Dako) or rabbit anti-mouse HRP diluted 1:2000 (Dako)) for 1 h at room temperature. Visualization of blots was done in a LAS 3000 imager (Fujifilm) with Amersham ECL Western Blotting Detection Kit (GE Healthcare). Post densitometric analysis and image processing of blots were performed in Image J.

Cells were lysed in a buffer consisting of 20 mM Hydroxyethyl piperazine Ethane Sulfonicacid (HEPES, pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The protein extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The blots were incubated with the appropriate primary and secondary antibodies and developed using an Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Science, Chicago, IL, USA).

Equal quantities of protein (30 µg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were blocked in 5% BSA at room temperature for 1 h. Then, the membranes were incubated with the following primary antibodies overnight at 4 °C: rabbit HDAC1, rabbit HDAC2, rabbit HDAC3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, 65816), rabbit IP3R (1:2000, Abcam, ab108517), rabbit p-CaMKⅡ (1:1000, Cell Signaling Technology, 12716), rabbit CaMKⅡ (1:1000, Proteintech, 13730-1-AP), rabbit ITPKA (1:1000, Proteintech, 14270-1-AP), and rabbit β-actin (1:2000, Proteintech, 20536-1-AP). The PVDF membranes were washed three times with 1× TBST and incubated with HRP-linked secondary antibody (Beyotime Biotechnology, Shanghai, China) at room temperature for 1 h. Finally, these membranes were incubated in an Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Sciences, Chicago, IL, USA) for 30 s–2 min, and the signals were visualized by exposure to Quantitative One Image Analysis (Bio-Rad, Hercules, CA, USA).

The protocol for Western blot was performed as previously described61 (link). Primary antibodies (anti α1 1:2000 (a6f-c, DevelopmenalStudies Hybridoma Bank), anti α2 1:1000 (07674, EMD Millipore, US), anti α3 1:1000 (06172, EMD Millipore, US), anti DAT 1:500 (MAB369, EMD Millipore, US), anti TH 1:2000 (AB152, EMD Millipore, US) and GAPDH 1:1000 (ab9485, Abcam, Cambridge, UK)) were incubated overnight at 4 °C. Next day, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (swine anti-rabbit 1:2000 (Dako, Glostrup, Denmark), rabbit anti-mouse 1:2000 (Dako, Glostrup, Denmark)) for 1 hour at room temperature. Visualization was done with a LAS 3000 imager (Fujifilm, Tokyo, Japan) using Amersham ECL Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK) as the detection reagent. ImageJ version 1.48 v was used for densitometric analysis of the Western blots.

To verify the impacts of AAV6-ADAMTS4 on the protein expression levels of ADAMTS4 and aggrecan western blot was applied. 1 x 10⁵ NP cells were seeded, transduced, cultured on the scaffold and harvested on day 8 and 48 as described before. For total protein isolation harvested cells were lysed for 20 min by using ice cold radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), which was supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Samples were centrifuged for 10 min (14000 × g, 4°C) and supernatants were used for western blot analysis. Total protein concentration in samples and controls was determined by BCA Protein Assay Kit (Thermo Scientific). Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Sigma-Aldrich) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Anti-ADAMTA4 antibody (SAB1411668, Sigma-Aldrich) and anti-aggrecan antibody (SAB4500662, Sigma-Aldrich) were used as primary antibodies. Interactions between antigens and primary antibodies were detected on the membrane by using horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (A0545, Sigma-Aldrich) and Amersham ECL Western Blotting Detection kit (GE Healthcare Life Sciences). Image J software was used to quantify protein bands.

Protein samples were prepared using RIPA buffer containing a protease inhibitor cocktail (Sigma Life Science and Biochemicals, St. Louis, MO, USA). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane as previously described [3 (link)]. Primary antibodies included Rabbit anti-human ATG5 (Cell Signaling technology, Beverly, MA, USA), mouse anti-HCV core (Fisher Scientific, Pittsburgh, PA, USA), and mouse anti-β-actin (Sigma, St. Louis, MO, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated enhanced chemiluminescence (ECL) donkey anti-rabbit IgG and HRP-conjugated ECL sheep anti-mouse IgG (GE Healthcare Biosciences, Pittsburgh, PA, USA). The blots were subjected to chemiluminescence assay using the Amersham ECL Western blotting detection kit (GE Healthcare Biosciences, Pittsburgh, PA, USA).