Tris glycine sds native sample buffer

MMP-9 activity in a medium derived from rhTGF-β1-treated MRPECs was determined by gelatin zymography. Briefly, the medium was mixed with Tris-Glycine SDS Native Sample Buffer (1:1; Invitrogen) and electrophoresed through 10% Novex Zymogram Gelatin Gels (Invitrogen) with Tris-Glycine SDS Running Buffer (Invitrogen) under constant voltage of 125 V for 120 min. The gel was then incubated in Zymogram Renaturing Buffer (Invitrogen) for 30 min at room temperature with gentle agitation and washed with developing buffer (Invitrogen) for 30 min. The gel was further incubated for 24 h in fresh developing buffer at 37°C. After being developed, the gel was stained with 0.5% (w/v) Coomassie Blue R-250 (Bio-Rad) in 50% (v/v) methanol, 10% (v/v) acetic acid for 30 min at room temperature, and destained as described previously [24 (link)]. Gelatinolytic activity of MMP-9 was visualized as a clear band on a blue background. Band intensity was quantified by densitometry using the ImageJ software. Briefly, zymogram gels were scanned using Kodak Gel Logic 100 imaging system and processed into gray scale. Gray scale images were quantified densitometrically by the measurement of the mean intensity of positive band multiplied by its corresponding area. The optical band intensity was then corrected by subtracting the background intensity of equal area.

At 48 hpi, the conditioned media from B19V-infected and mock-infected NHDFs were collected, centrifuged, diluted with Tris–Glycine SDS Native Sample Buffer (Invitrogen, Italy) and loaded on 10% Novex Zymogram Gelatine Gels (Invitrogen, Italy). The gels were developed following manufacturer’s instructions and quantified using ImageJ software.