Gs 710 imaging densitometer

GS-710 Imaging Densitometer (Bio-Rad) and PDQuest software (version 4.7.0, Bio-Rad) were used to capture, store, and analyze protein spots on 2D-E gels and lectin blots. PDQuest software matched the identical spots in a series of gels and normalized the gels to compensate for any variations between gels, especially those caused by varying experimental conditions. The analysis was normalized by total density in gel, which accounts for the raw quantity of each spot in a gel, divided by the total intensity value of all the pixels in the image. The normalized spot quantity was expressed as percentages of volume contributions (vol%) to facilitate the data compilation. Data was checked manually to eliminate possible error in matching pairs.

2D-PAGE was done essentially as previously described [49 (link)]. Six replicates of each cell line were harvested and from each sample 75 µg of total protein was loaded on the gels. In short, first dimension isoelectric focusing (IEF) was performed using pH 3–10 nonlinear 18 cm strips (GE Healthcare). Separation in the second dimension was performed using polyacrylamide gels (12% T, 3% C). Gels were then silver stained and scanned on a GS-710 Imaging Densitometer (Bio-Rad, Hercules, CA, USA) using Quantity-One. The TIFF files were then imported into PDQuest software (BioRad, Hercules, CA, USA). Protein spots were initially automatically defined and quantified and then manually investigated for proper alignment between the gels. Differentially expressed spots were defined as spots that were 2-fold or more differentially expressed with a significance level at p < 0.05 (Mann-Whitney U-test).

Lung tissue samples from the Control group and Experimental group (48 h post-exposure) were crushed using liquid nitrogen and suspended in lysis buffer (9.5 M urea, 4% CHAPS, 65 mM DTT, and 0.2% carrier ampholyte) which contained a protease inhibitor cocktail (Roche, Mannheim, Germany). The suspension was homogenized, sonicated on ice and centrifuged at 14,000 rpm for 1 h at 4 °C. The supernatant was collected and the protein concentration was determined using a Protein Assay kit (Bio-Rad, Richmond, VA, USA). Protein aliquots equivalent to 100 mg were stored at −80 °C for future use.
2-DE (Bio-Rad) was used to separate proteins as described by De-la-Pena C et al. [32 (link)]. The 2-DE gels were scanned using a GS-710 imaging densitometer and the digitized images were analyzed with the PDQuest software (Bio-Rad).

One milligram of protein extracts was prepared from about 3 × 10⁸ trophozoites arrested with aphidicolin (G1/S-phase cells) and trophozoites released from the aphidicolin-mediated arrest (G2-phase cells) by resuspending them in a 2-DGE sample buffer (7 M urea, 2 M thiourea, 100 mM DTT, 4.5% CHAPS and 40 mM Tris). Immobilized pH gradient (IPG) gel strips (pH 3–10, 18 cm; GE Healthcare, Uppsala, Sweden) were soaked overnight with the extracts. The rehydrated IPG strips were treated for sequential isoelectric focusing at 80 kV. Second-dimension separation was carried out at room temperature on 9–17% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels (20 × 25 cm). The protein bands on the gel were visualized by Coomassie staining. The 2-DGE experiment was performed twice. Stained gels were scanned using a GS-710 imaging densitometer (Bio-Rad, Hercules, CA, USA) and analyzed with Melanie 5 image analysis software (GE Healthcare). Image labeling was processed using Adobe Photoshop v.7.0 software. Analysis of these two-dimensional (2-D) gels was performed at Yonsei Proteome Research Center (Seoul, Korea).