Biorad criterion tgx

FPLC was performed as previously described^{40 (link)}. Serum (50 µL) from two representative noncarrier or carrier siblings was injected into an ÄKTA Go FPLC (GE Healthcare). A Superdex 200 10/300 GL column (GE Healthcare) was used to separate adiponectin complexes in HEPES/Ca²⁺ buffer (25 mM HEPES; 150 mM NaCl; and 1 mM CaCl₂, pH 7.4). 250 µL fractions were collected over a 20 mL retention volume. The retention volumes found to contain adiponectin were then used to run Western blots to determine HMW, LMW and trimeric adiponectin. Samples were run on an SDS-Page gel (BioRad Criterion TGX) after being reduced in Laemmli and 355 mM 2-mercaptoethanol and boiled for 10 min. The gel was transferred to PVDF membrane (BioRad). The membranes for each patient were blocked in 5% BSA then probed for adiponectin overnight with rabbit polyclonal anti-adiponectin at a 1:1000 dilution (Abcam, ab75989). There were washed then stained with goat-anti-rabbit AlexaFluor Plus 680 (Invitrogen) at a 1:4000 dilution. Then washed again and imaged on a ThermoFisher iBright system. Western blots derive from the same experiment and were processed in parallel.

Samples were normalized by OD₆₀₀ values (whole cell lysates), protein concentration (subcellular fractionation samples), or colony forming units (CFU/mL; murine vaginal washes). Proteins were separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% Novex NuPAGE (Thermo Fisher Scientific), 4–15% Bio-Rad Criterion TGX (Bio-Rad), or Bio-Rad AnykD Criterion TGX (Bio-Rad) gels and visualized by colloidal Coomassie G-250 staining. Immunoblots were performed as described (Zielke et al., 2016 (link); Sikora et al., 2017 (link), 2018 (link); Wierzbicki et al., 2017 (link)). The polyclonal rabbit anti-Laz antiserum used in the current study was generated previously by immunization with recombinant His-tagged Laz (Zielke et al., 2016 (link)).