Low fat milk

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.

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.