Western blot analysis of protease-activity in wild type N. tabacum leaf extracts of different pH. Human IgG1κ was mixed with wild-type N. tabacum leaf extracts buffered at pH 3.0, 4.2, 5.0, 6.2, 7.4 or 8.0 and incubated at room temperature for 15 mins (Panel A); 2 hrs (Panel B); or 24 hrs (Panel C). Proteins were separated by 6% SDS-PAGE under non-reducing conditions. The proteins were blotted onto nitrocellulose and probed with anti-human γ1 (Fc region specific) antiserum. PC is the positive control human IgG1κ (1.8 ng/lane). For three of the pH conditions, the effect of a protease inhibitor cocktail was tested (± PI, Panels D and E). Samples were tested at 24 hrs and immunodetection was with anti-heavy chain antiserum (Panel D) or anti-light chain antiserum (Panel E). The asterisk indicates the fully assembled 2G12 antibody; lower case letters (a, b and c) indicate antibody fragments.
Hehle et al. BMC Biotechnology 2011 11:128 doi:10.1186/1472-6750-11-128