cona agarose beads  (Millipore)

Journal: The Febs Journal

Article Title: The molecular species responsible for α 1 ‐antitrypsin deficiency are suppressed by a small molecule chaperone

doi: 10.1111/febs.15597

Figure Lengend Snippet: Identification of polymers in the soluble and insoluble intracellular fractions of cells expressing Z α 1 ‐antitrypsin. (A) CHO‐inducible cells expressing either M or Z α 1 ‐antitrypsin were induced with 0.5 μg·mL −1 doxycycline for 48 h and then lysed in 1% v/v NP‐40 buffer. The soluble (Sol), insoluble (Insol) and extracellular (EC) fractions were separated by 4–12% w/v acrylamide SDS/PAGE and the proteins visualised by immunoblotting for total α 1 ‐antitrypsin with the 3C11 mAb. White and black arrowheads indicate the mature and immature glycosylated forms of α 1 ‐antitrypsin, respectively. The percentage of intracellular and secreted α 1 ‐antitrypsin was calculated by densitometric quantification. Graph shows mean ± standard error of the mean (± SEM, n = 3). (B) Cell lysates from CHO cells expressing either M or Z α 1 ‐antitrypsin (as indicated) were concentrated with ConA beads and eluted as described in Methods. The samples were then resolved on a 3–12% w/v acrylamide nondenaturing gel followed by immunoblotting for total α 1 ‐antitrypsin. White and black arrowheads indicate the mature and immature glycosylated forms of α 1 ‐antitrypsin, respectively. The different charges acquired from the glycosylation process make the mature form of the protein run faster than the immature form. Monomeric secreted Z α 1 ‐antitrypsin in the supernatant migrates more slowly in nondenaturing PAGE than secreted M α 1 ‐antitrypsin as a result of the Glu342Lys mutation. The immunoblot is representative of 3 independent experiments. (C) 1% v/v NP‐40‐soluble and NP‐40‐insoluble fractions from cells expressing either M or Z α 1 ‐antitrypsin were sequentially immunoprecipitated using the 2C1 polymer‐specific mAb (three times) and the 3C11 mAb against total α 1 ‐antitrypsin (once) as indicated for Set 1. The fractions were also immunoprecipitated three times with the 3C11 mAb and once with 2C1 mAb as shown in Set 2. Proteins were eluted with an SDS‐based buffer and analysed by 4–12% w/v acrylamide SDS/PAGE followed by immunoblotting for α 1 ‐antitrypsin with polyclonal antibody. Western blots are representative of two independent experiments.

Article Snippet: For analysis in nondenaturing PAGE, total α 1 ‐antitrypsin from cell lysates and culture medium samples was concentrated using ConA‐conjugated agarose beads (Sigma‐Aldrich Co, Dorset, UK).

Techniques: Expressing, SDS Page, Western Blot, Mutagenesis, Immunoprecipitation