Analysis of recombinant NtMMP1 produced transiently in tobacco leaves. A: Immunoblot analysis of fractions from immobilized metal affinity chromatography purification of NtMMP1-apo. Equal volumes of the different fractions were separated by 12% (w/v) SDS PAGE, blotted onto nitrocellulose membranes and probed with a Penta-His antibody (Qiagen) diluted 1:5000, followed by detection with a goat anti-mouse AP-labeled Fc-specific antibody (Dianova) diluted 1:10.000 and development with NBT/BCIP. Lane 1: protein extract from wild type plants; 2: flow through fraction; 3: wash fraction; 4–6: elution fractions. B: Zymography of recombinant NtMMP1 (NtMMP1-apo and NtMMP1-KDEL). Equal amounts of NtMMP1-apo and NtMMP1-KDEL were separated by 12% (w/v) SDS PAGE containing 0.1% (w/v) casein. Lane 1: NtMMP1-KDEL with APMA treatment; 2: NtMMP1-KDEL without APMA treatment; 3: NtMMP1-apo with APMA treatment; 4: NtMMP1-apo without APMA treatment. C: Zymography in the presence of 10 mM EDTA. Samples were applied as listed in B.