Extracted protein (3 μg mL−1) was stained with 4X lithium dodecyl sulphate (LDS) sample buffer and subjected to sodium dodecyl sulfate (SDS)—polyacrylamide (12%) gel electrophoresis run at 120 V. Upon completion, the gel was stained in Coomassie Blue staining solution until a clear background was obtained for scanning with GS-800 calibrated densitometer (Bio-Rad, USA). In western blot analyses, electrophoresed gels were transferred to BioTrace NT nitrocellulose transfer membrane (Pall, USA). Membranes were incubated overnight at 4°C in gelatin from cold water fish skin (blocking agent) (Sigma, USA). The production of PBP2a from MRSA was detected by probing the membranes with mouse anti-PBP2a primary antibody (Denka Seiken, Japan) and antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Thermo Scientific, USA) with a dilution factor of 1 : 10000. The same membranes were hybridized with anti-mouse horseradish peroxidase-linked secondary antibody (Abcam, UK) diluted to 1 : 10000 to facilitate colorimetric detection with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Nacalai Tesque, Japan). Assay response was recorded using GS-800 calibrated densitometer (Bio-Rad, USA). Densitometric quantification of western blot images was done using Image J 1.38 programme (Windows version of NIH Image). Results were scored in percentage of expression (%) normalized to GAPDH control.
Gs 800 calibrated densitometer
Manufactured by Bio-Rad
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The GS-800 Calibrated Densitometer is a lab equipment designed for scanning and quantifying optical density in a variety of sample types, including electrophoretic gels and blots. It provides precise and accurate measurements to support data analysis in various research and diagnostic applications.
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Lab products found in correlation
Quantity one software, by Bio-Rad (27 mentions)
Quantity one, by Bio-Rad (6 mentions)
Hybond, by Cytiva (6 mentions)
Pdquest software, by Bio-Rad (6 mentions)
Quantity one v4, by Bio-Rad (5 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Protean ief cell, by Bio-Rad (3 mentions)
Readystrip ipg strip, by Bio-Rad (3 mentions)
Pdquest 8, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Pdquest 2 d analysis software, by Bio-Rad (3 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (3 mentions)
Nitrocellulose, by Cytiva (3 mentions)
Whatman, by Cytiva (3 mentions)
Amersham hyperfilm ecl, by GE Healthcare (3 mentions)
Mini protean 3 electrophoresis cell unit, by Bio-Rad (3 mentions)
Protein assay dye, by Bio-Rad (3 mentions)
Las 4000, by Fujifilm (3 mentions)
Coomassie brilliant blue r 250, by Merck Group (3 mentions)
Immobilon p, by Merck Group (3 mentions)
229 protocols using gs 800 calibrated densitometer
1
SDS-PAGE and Western Blot Analysis
2
Quantitative Analysis of LukF-PV and Hla
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Trichloroacetic acid was added to culture supernatants and incubated at 4°C overnight. Precipitates were then harvested by centrifugation, washed with acetone, air-dried and solubilized in a SDS and 2-mercaptoethanol containing sample buffer. The proteins were separated on 12% SDS-PAGE. A peptide sequence specific to LukF-PV, HWIGNNYKDENRATHT was synthesized and HRP conjugated polyclonal chicken IgY was raised against this peptide (Genscript). This antibody was used to detect LukF-PV with enhanced chemiluminescence. Images generated from the western blots were quantitated using GS800 Calibrated Densitometer (BioRad) and Image J
[32 ].
Hla was detected using a polyclonal rabbit anti-Hla (Sigma-Aldrich), in buffer containing 20 mM DEPC to inhibit non-specific protein A binding and HRP conjugated sheep anti-rabbit secondary antibody (Millipore) with enhanced chemiluminescence detection
[33 (link)]. For comparison of JKD6159 versus international clone collection (Table
1 ), images generated from the Hla western blots were quantitated using GS800 Calibrated Densitometer (BioRad) and Image J
[32 ].
Subsequently, for comparison of JKD6159 and other ST93 strains (Table
1 ), detection of chemiluminescence was performed using the MF-ChemiBIS 3.2 platform (DNR Bioimaging systems). Quantitation was performed using Image J
[32 ].
[32 ].
Hla was detected using a polyclonal rabbit anti-Hla (Sigma-Aldrich), in buffer containing 20 mM DEPC to inhibit non-specific protein A binding and HRP conjugated sheep anti-rabbit secondary antibody (Millipore) with enhanced chemiluminescence detection
[33 (link)]. For comparison of JKD6159 versus international clone collection (Table
[32 ].
Subsequently, for comparison of JKD6159 and other ST93 strains (Table
[32 ].
3
Tumor Volume Quantification and Statistical Analysis
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The tumor volume of individual mice during treatment was divided by starting volume to calculate relative change in tumor volume, and linear regression was used to model tumor growth rate. Student’s t test was used to compare continuous variables. All group comparisons were unpaired. Continuous variables were expressed as means ± SEM. All P values reported are two-tailed, and statistical significance was indicated by P values of < .05. GraphPad Prism (Version 5.0b) software (La Jolla, CA) was used for all statistical analyses. Image acquisition and analysis for all Western blot data were performed using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) and Image Studio V2.1 software (LI-COR) and for all array data using a Bio-Rad GS-800 calibrated densitometer (Bio-Rad, Hercules, CA) and ImageQuant TL 2005 (GE Healthcare, Piscataway, NJ) software. Blots and array images were cropped and spliced for clarity and ease of comparison using Adobe Photoshop CS4 (Adobe, San Jose, CA).
4
Omega Fatty Acid Protein Expression
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Cells were treated with different ratios of omega fatty acids for 24 h. Then, total cellular protein was extracted using 1% Triton X-100 in PBS containing a protease inhibitor cocktail tablet and the protein concentration was determined by using Bio-Rad protein assay kit, as per manufacturer's instructions (Bio-Rad Laboratories, United Kingdom). Protein (20-40 µg) was separated on 10% SDS-PAGE gels (Thermo Scientific Pierce, United Kingdom) by electrophoresis for 90 min at 120 V and transferred onto a nitrocellulose membrane (0.45 µm) for 1 h at 350 A. Membranes were blocked with 1% BSA, except when confirming β-actin where 5% BSA was used. The proteins examined were SCD1 (1:4000) (Abcam, United Kingdom), PPAR-α (1:2000) (Abcam, United Kingdom), CB1, (1:4000) (Santa Cruz Biotechnology, United Kingdom) CB2 (1:2000) (Abcam, United Kingdom), and β-actin (1:4000) (Abcam, United Kingdom). West PICO Chemiluminescent Substrate (ThermoFisher, United Kingdom) was used for detection and the X-ray films were scanned on a Bio-Rad GS-800 Calibrated Densitometer (Bio-Rad, United Kingdom)[23 (link),24 (link)]. During post-densitometry analysis, data were expressed relative to corresponding β-actin levels and compared to the experimental control.
5
Western Blot Analysis of PARP and Caspase-3
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PolyADP-ribose polymerase (PARP) and caspase-3 cleavage were assessed using a standard western blotting protocol [17] (link). Membranes were probed using polyclonal rabbit anti-PARP1 antibody (Abcam, Cambridge, Cambridgeshire, UK; 1∶1000), a rabbit monoclonal anti-ERCC1 antibody (Cell Signaling Technology, Danvers, MA, USA; 1∶1000) and a polyclonal rabbit anti-cleaved caspase-3 antibody (Cell Signaling Technology; 1∶1000). A rabbit anti-actin antibody (Sigma-Aldrich Co. Ltd; 1∶2000) was used as a loading control. Blots were scanned (Bio-Rad GS-800 Calibrated Densitometer; Bio-Rad, Hercules, CA, USA) and signal quantification was performed by densitometry using scanning analysis software (Quantity One; Bio-Rad).
6
Skin Tissue Protein Analysis
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Skin tissues from patients were homogenized and sonicated in lysis buffer (containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% Triton, 0.1% sodium dodecyl sulfate (SDS) and 1% protease inhibitors). Equal amounts of proteins were separated on 7–12% SDS-polyacrylamide gels in a minigel apparatus (Bio-Rad, Hercules, CA, USA) and then transferred electrophoretically to nitrocellulose membranes, followed by blocking with milk and incubation at 4°C overnight with anti-K10, LOR, FLG, TNF-α and IL-8 Abs, as well as anti-p65, phosphorylated-(p-)p65, inhibitor κBα (IκBα) and p-IκBα (phosphorylated at Ser32) Abs (Cell Signaling Technology, Inc.). Membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary Abs. Subsequent to immunoblotting, the films were scanned and the intensity of the immunoblotting bands was detected with a Bio-Rad GS-800 Calibrated Densitometer (Bio-Rad). GAPDH and α-tubulin were used as the loading controls.
7
Long-term Proliferation Assay of PRDX1-downregulated Cells
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To evaluate the long-term proliferation rate of PRDX1-downregulated MDA-MB-231 cell line, modified and control cells were plated in pre-tested appropriate densities yielding 1000 into 6-well culture plates and cultured for seven days to allow colony formation. Next, the colonies were stained with 0.5% crystal violet (Sigma-Aldrich) in 20% methanol. Digital images of the colonies were obtained using a BioRad GS-800 Calibrated Densitometer (BioRad Laboratories, Hercules, CA, USA) and further analyzed with Fiji software [31 (link)]. Colonies smaller in size than 13 square pixels were omitted from the analysis according to the digital analysis guidance for breast cancer cell lines [32 (link)]. Survival fraction was plotted as the percent of the plating efficiency of shNTC and shPRDX1 MDA-MB-231 in comparison to parental MDA-MB-231 cells. The experiment was performed in triplicates and repeated three times. Additionally, the number of cell colonies, their area, and the distribution of their size was calculated and plotted with the help of ImageJ macro PHICS (Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland) [33 (link)].
8
Proteomic Analysis of Biological Samples
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The gels were scanned using a GS-800™ Calibrated Densitometer (Bio-Rad, Hercules, CA, USA) and spots were detected and matched with the aid of a PDQuest Analysis software version 8.0.1 Advanced (Bio-Rad, Hercules, CA, USA). Protein spots were detected and manually landmarked to the master gel (that displayed the highest number of spots) to improve matching quality before the automatic matching. Following this step, only the spots that were present on at least six gels were further processed. The area-based method was used as the parameter for spot quantification after local regression (LOESS) normalization. The degree of difference between protein groups was expressed as an average ratio. To measure the variability within the group, the coefficient of variation was calculated for each experimental replicates. For the statistical analysis of the differences in relative abundance of protein spots Student’s t-test was used as integrated in the PDQuest software. Significance of the differences was set at the level of p ≤ 0.05. The experimental molecular masses were assessed using Precision Plus Protein™ Kaleidoscope™ Standard for SDS-PAGE (Bio-Rad, Hercules, CA, USA).
9
Western Blot Analysis of HSP90 Protein
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Each sample was mixed with 5× sample loading buffer and boiled at 95°C for 8 min, and was separated on a 10% SDS-PAGE gel, then transferred onto nitrocellulose membranes using 200 mA of constant current. Membranes were then incubated with primary antibodies specific (HSP 90α/β, Santa Cruz Biotechnology, SC-59578, Achlya Ambisexualis Origin, Monoclonal, USA) for target proteins for 2 h at room temperature. Subsequently, membranes were washed three times with TBST. Then membranes were incubated with secondary antibody (Anti-Mouse produced in rabbit, SAB3701212, Sigma) for 2 h at room temperature followed by the same wash step. Membranes were visualized with ECL Western Blotting Substrate. β-Actin was used as an internal reference protein to normalize protein expression. The images were scanned with a GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, USA), and the lanes were analyzed with Image J software.
10
Two-Dimensional Gel Electrophoresis Protocol
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Samples (50 μg protein) were diluted in 100 μl of IEF buffer [8 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (v/v) Triton X-100, 50 mM dithiothreitol], 3 μl of ampholytes were added, and loaded onto 7 cm IPG strips of pH 3–10 (ReadyStripTM IPG Strips BioRad) for IEF. Isoelectric focusing was carried out using Protean IEF Cell (Bio-Rad) with the following conditions: 150 V for 150 VH, 500 V for 500 VH, and 4000 V for 15,000 VH including initial active rehydration for 12 h at 50 V. For the second dimension (SDS-PAGE), IPG strips were incubated in SDS equilibration buffer [1.5 M Tris-HCl pH 6.8, 6 M urea, 30% (v/v) glycerol, 5% (w/v) SDS) for 15 min with 2% (w/v) dithiothreitol] followed by a second equilibration step of 15 min with the equilibration buffer containing 2.5% (w/v) iodoacetamide. The equilibrated strips were loaded on the top of 10% polyacrylamide gel and the electrophoresis was run at 80 V until the dye reached the bottom of the gel. Gels were stained for 16 h with Coomassie Brilliant Blue G-250 at room temperature. The 2-DE gels were digitalized using a GS-800 Calibrated Densitometer (Bio-Rad) at 300 dpi and 16 bit grayscale. Digitalized gels were analyzed with PDQuest 8.0 software (Bio-Rad).
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