Journal: BMC Biotechnology

Article Title: Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha

doi: 10.1186/1472-6750-12-96

Figure Lengend Snippet: a. Western blot analysis of supernatants from rSAK producing strains. Lane 1 THR 174 ~ 60ng; RB11/pFPMT Mfα + - rSAK-1 (lane 2 – 10) and RB11/pFPMT Mfα + - rSAK-2 (lane 11–18); 7 μL loaded. b : Glycosylation evidence for SAK: SDS-PAGE analysis of de-repression supernatants. (Pool 1) was de-repressed in YP 2% glycerol for 40 h at 30°C and 37°C. Pooled supernatants of pool 1 were treated with EndoH (lane 4 and 6). Supernatants without EndoH treatment (lane 3 and 5) have been handled with the same EndoH buffers but omitting EndoH. After EndoH treatment, samples were prepared for SDS PAGE and 21 μL each was loaded on a Criterion 4-20% precast gradient gel. Supernatants have been finally examined in a Western blot analysis for presence of SAK. SAK protein was detected by using alkaline phosphatase-conjugated mouse anti-SAK antibody. Approximately 180 ng of THR174 served as positive control.

Article Snippet: Voltage was adjusted to 150 V. The following precast gels were used for the expression studies: 4–20% Criterion precast gradient gel (BioRad #354-0032) in a Criterion-Cell (BioRad).

Techniques: Western Blot, Glycoproteomics, SDS Page, Positive Control