After binding of the DNA origami blocks on the bilayers and overnight incubation, we imaged the samples under various buffer conditions.
Aliquots were taken after 24 h, and samples were prepared with NuPAGE LDS sample buffer under reducing conditions.
Under the non-reducing condition, samples were mixed with sample buffer lacking β-mercaptoethanol and boiled.
Protein samples were mixed with sample buffer, boiled for 10 min, separated by SDS-PAGE under denaturing conditions, and electroblotted to nitrocellulose membranes.
Lysates were boiled 5 min with SDS sample buffer under reducing conditions, resolved by SDS-PAGE and transferred to nitrocellulose membranes by electroblotting.
For western analysis of MMP-2, concentrates of conditioned medium containing 2 μg protein were mixed with 5X Laemmli sample buffer under nonreducing conditions at room temperature for 10 min, and separated in 10% polyacrylamide gels by SDS-PAGE.
Having established that the TSP-5-1C proligomerizeomerize and are secreted effectively, ECM was prepared from transfected COS7 cells and extracted in hot SDS/PAGE sample buffer under reducing conditions [ 20].
Gelatin zymography was performed using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, 7.5%) that was copolymerized with 1 mg/mL gelatin type B (Sigma, St . Louis MO, USA), and the supernatants were diluted in SDS-PAGE sample buffer under nonreducing conditions without heating.
In this type of assay, the reactions are terminated under reducing conditions (SDS sample buffer with DTT) or under non-reducing conditions (SDS sample buffer with 8 M urea).
Protein standards and samples were prepared under reducing conditions with Laemmli sample buffer for 5 minutes at 95°C or under non reducing conditions (Laemmli sample buffer without reducing agent) for 10 min at 65°C.
