Primary neurons were lysed in RIPA buffer and extracted proteins were mixed with Laemmli buffer to prepare them for electrophoresis. Zebrafish embryos were directly lysed in Laemmli buffer at 95 °C for 15 min. Proteins bands from lysates were resolved by SDS-PAGE using a 4% stacking gel and 6 or 7.5% resolving gels. Then, proteins were transferred to PVDF membranes with a semi-dry transfer system and visualized by ECL (Bio-Rad) detection. The protein bands obtained by ECL using film exposures in the linear range were imaged using a GS-900 calibrated imaging densitometer (Bio-Rad) and quantified using Image Lab Software (Bio-Rad). Standard errors were calculated after densitometry from at least three separate experiments.
Gs 900 calibrated imaging densitometer
Manufactured by Bio-Rad
The GS-900 calibrated imaging densitometer is a laboratory instrument designed for the quantitative analysis of gel-based assays, including Western blots, gel electrophoresis, and other gel-based techniques. The device captures high-quality images of these samples and provides precise measurements of band or spot intensity for subsequent data analysis.
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5 protocols using gs 900 calibrated imaging densitometer
1
Western Blot Analysis of Proteins
2
Separation and Characterization of RBD Proteins
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RBD(319–541)-HEK_A3, (RBD(319–541)-CHO)2, and RBD(331–529)-Ec proteins were separated by SDS-PAGE as described by Laemmli [22 (link)], under reducing and non-reducing conditions. Two micrograms of N-glycosylated and deglycosylated proteins were applied in a 12.5%T, 3%C acrylamide-bisacrylamide separating gel at 30 mA/gel until the tracking dye left the gel. Proteins were detected by silver staining [23 (link)] or Coomassie Brilliant Blue G-250; gel images were analyzed with a GS-900 calibrated imaging densitometer (Bio-Rad) and processed with Image Lab v6.0 software (Bio-Rad).
3
Quantifying Protein Expression by ECL
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The protein bands obtained by ECL (Bio-Rad) using film exposures in the linear range were imaged using a GS-900 calibrated imaging densitometer (Bio-Rad) and quantified using Image Lab Software (Bio-Rad). All statistical analyses were performed using GraphPad Prism (GraphPad Software). Kruskal-Wallis was used to compare multiple groups, with subsequent Dunn’s post-hoc test to determine the significant differences between samples. Kolmogorov-Smirnov and Mann-Whitney U tests were used to compare two separate groups. p values are denoted through the text as follows: *p < 0.05; * *p < 0.01; ** *p < 0.001; ** **p < 0.0001; p < 0.05 or lower values were considered significantly different when compared by one-way Kruskal-Wallis (Dunns’s posthoc test) or Kolmogorov-Smirnov and Mann-Whitney U tests. Thorough the text, the Box whisker plots represent median (line), mean (point), 25–75 percentile (box), 10–90 percentile (whisker), 1–99 percentile (X) and min - max (−) ranges.
4
Western Blot Analysis of Glycine Transporters
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Protein samples were separated by SDS-PAGE using a 4% stacking gel and 6 or 7.5% resolving gels. The samples were transferred to nitrocellulose with a semi-dry transfer system (Life Technologies Inc.: 65 V/gel, 90 min). Membranes were blocked for 30 min with 5% milk in PBS containing 0.1% Tween 20 at 25 • C. The membranes were probed overnight at 4 • C with the desired primary antibody: anti-GlyT2 (rabbit 1:1000); anti-GlyT2 (rat, 1:500); anti-Myc (mouse, 1:1000) or antiubiquitin (mouse, 1:200). After several washes, the antibodies bound were detected with peroxidase coupled anti-rat (1:8000; Sigma), antirabbit (1:8000; Bethyl) or anti-mouse IgG (1:8000; ThermoFisher Scientific) which were visualized by enhanced chemiluminescence (ECL; Amersham Corp.). Subsequently, the antibodies were stripped from the membrane (Thermo Scientific), which was re-probed with anti-tubulin (mouse, 1:2000), anti-CNX (mouse, 1:1000); anti-transferrin receptor (mouse, 1:500) or anti-GlyT2 (rat 1:500) as loading controls. Antibody binding was detected with a peroxidase-coupled anti-mouse or anti-rat IgG. Protein bands visualized using film exposures in the linear range were imaged using a GS-900 calibrated imaging densitometer (Bio-Rad) and quantified using Image Lab Software (Bio-Rad).
5
Western Blot Protein Expression Analysis
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Protein samples were separated by SDS-PAGE using a 4% stacking gel and 6 or 7.5% resolving gels. The samples were transferred to nitrocellulose with a semi-dry transfer system (Life Technologies Inc.: 65V/gel, 90 min). Membranes were blocked for 30 min with 5% milk in PBS containing 0.1% Tween 20 at 25 °C. The membranes were probed overnight at 4 °C with the desired primary antibody: anti-GlyT2 (rabbit 1:1,000); anti-GlyT2 (rat, 1:500); anti-Myc (mouse, 1:1,000) or anti-ubiquitin (mouse, 1:200). After several washes, the antibodies bound were detected with peroxidase coupled anti-rat (1:8,000; Sigma), anti-rabbit (1:8,000; Bethyl) or antimouse IgG (1:8,000; ThermoFisher Scientific) which were visualized by enhanced chemiluminescence (ECL; Amersham Corp.). Subsequently, the antibodies were stripped from the membrane (Thermo Scientific), which was re-probed with anti-tubulin (mouse, 1:2,000), anti-CNX (mouse, 1:1,000); anti-transferrin receptor (mouse, 1:500) or anti-GlyT2 (rat 1:500) as loading controls. Antibody binding was detected with a peroxidase-coupled anti-mouse or antirat IgG. Protein bands visualized using film exposures in the linear range were imaged using a GS-900 calibrated imaging densitometer (Bio-Rad) and quantified using Image Lab Software (Bio-Rad).
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