Lf pvdf membranes

Material obtained from IP or ConA‐agarose concentration as described in the previous section was resolved either by SDS/PAGE or by nondenaturing PAGE on pre‐cast NuPAGE™ 4–12% w/v acrylamide Bis/Tris Protein Gels and NativePAGE™ 3–12% w/v acrylamide Bis/Tris Protein Gels (Bio‐Rad Laboratories Ltd), respectively. Samples were then transferred to LF‐PVDF membranes (Millipore Ltd, Hertfordshire, UK). Membranes were saturated in 5% w/v low‐fat milk (Cell Signaling Technology, Danvers, MA, USA) (New England Biolabs Ltd) in PBS‐0.1% v/v Tween, probed with the indicated primary antibodies and horseradish peroxidase (HRP)‐conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, USA) and revealed by ECL (Clarity; Bio‐Rad Laboratories Ltd). Western blot images were acquired with the Image Quant Las400 (GE Healthcare Life Sciences, CA, USA) and analysed with image studio lite software (LI‐COR Biosciences, Cambridge, UK). Statistical analysis was performed using the graphpad prism program.

EDL muscles were secured to approximate in situ length and incubated in either standard PSS or 1.2% BaCl₂ in PSS for 1 h at 37 °C then frozen in liquid nitrogen. Following homogenization, protein concentration of the supernatant was quantified with the Bradford method (Cat. # 5000006; Sigma). Protein concentration of each sample was normalized in 4x Laemmli sample buffer (Cat. # 1610747, Bio-Rad; Hercules, CA, USA) containing 5% dithiothreitol. Samples were loaded on 4–20% gradient Mini-Protein TGX gels (Bio-Rad) for electrophoresis and transferred to LF-PVDF membranes (Millipore; Burlington, MA). Following 2 h blocking in 5% milk, membranes were incubated overnight at 4 °C and again for 3 h at 25 °C in primary antibody raised against αII-spectrin (1:250, Cat. # sc48382, Santa Cruz; Dallas, TX, USA). A secondary antibody (Alexa Fluor 800 IgG, 1:5000; Cat. # 926–32,212, Li-Cor Biosciences; Lincoln, NE, USA) was used to quantify protein differences with an Li Cor Odyssey Fc imaging system. Western blots were normalized to total protein according to the recommendations for fluorescent Western blotting [22 (link)] using Revert total protein stain (Cat. # 926-11010, Li-Cor). The 40 kDa bands correlate with the total protein in each lane and are shown to represent equal protein loading [23 (link), 24 (link)].

Samples analyzed by SDS-PAGE were resolved on pre-cast NuPAGE™ 4–12% w/v acrylamide Bis/Tris Protein Gels (Invitrogen, Thermo Fisher Scientific Ltd., Loughborough, UK) and transferred to LF-PVDF membranes (Millipore Ltd., Hertfordshire, UK). Membranes were saturated in 5% w/v low-fat milk (Cell Signaling Technology, Danvers, MA, USA) in PBS-0.1% v/v Tween, probed with anti-human AAT polyclonal antibody (Dako, Agilent, Stockport, UK) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), and finally revealed by ECL (Clarity; Bio-Rad Laboratories, Watford, UK). Western blot images were acquired with an Image Quant Las400 (GE Healthcare Life Sciences, Amersham, UK) and analyzed using Image Studio Lite software (LI-COR Biosciences, Cambridge, UK). For non-denaturing PAGE, we followed a procedure previously reported for serpin polymers [51 (link),59 (link)]. Briefly, the samples were mixed with non-denaturing loading buffer (without β-mercaptoethanol and SDS), analyzed with 7.5% w/v acrylamide gels made in-house, and transferred to PVDF membranes in the absence of methanol, followed by immunoblot as described above.