After the electrophoresis, the resulting gel was visualised using an image analysis system (UVITEC Cambridge, United Kingdom). The images were photographed with a digital camera (Kodak, Japan), as shown in Figure 1 , to detect the BlaCTXM-1 gene.
Image analysis system
Manufactured by Uvitec
Sourced in United Kingdom
The image analysis system is a device designed to capture, process, and analyze digital images. It provides core functionalities for image acquisition, enhancement, measurement, and data extraction. The system utilizes advanced algorithms and software to extract relevant information and metrics from the captured images.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Digital camera, by Kodak (1 mentions)
Luminata crescendo, by Merck Group (1 mentions)
Nucleospin rna protein, by Macherey-Nagel (1 mentions)
Hybond c nitrocellulose membranes, by Thermo Fisher Scientific (1 mentions)
Anti human αsma, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Superblock blocking buffer in pbs, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
3 protocols using image analysis system
1
Visualizing BlaCTXM-1 Gene Expression
2
Quantitative Protein Expression Analysis
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Proteins were extracted using NucleoSpin RNA/protein (Macherey-Nagel) and quantified by the Bicinchoninic acid method. For each experimental condition, 30 μg of proteins were separated by electrophoresis on Bis-Tris gel (GenScript Biothec, Leiden, Netherlands) and transferred onto Hybond-C-nitrocellulose membranes (Life Technologies Ltd. Paisley, UK).
After 1 h in blocking solution (SuperBlock Blocking buffer in PBS, Thermo Scientific), membranes were incubated overnight at 4 °C with the following primary antibodies: anti-human αSMA (dilution 1:300, Sigma-Aldrich), S100A4 (dilution 1:100, Santa Cruz Biotechnology, Dallas, USA), COL1 (dilution 1:600, Proteintech), and FN (dilution 1:2,000, Sigma-Aldrich). The membranes were subsequently incubated with (HRP)-conjugated secondary antibodies (dilution 1:2,000; Cell Signaling, MA, USA). To confirm similar loading of protein samples into the gels and the efficiency in the electrophoretic transfer, membranes were incubated with primary HRP-conjugated antibody to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:2,000; Santa Cruz Biotechnology). Protein synthesis was detected using the enhanced chemiluminescence system (Luminata Crescendo, Millipore), and the densitometric analysis was performed by the UVITEC Image Analysis System (UVITEC, Cambridge, UK).
After 1 h in blocking solution (SuperBlock Blocking buffer in PBS, Thermo Scientific), membranes were incubated overnight at 4 °C with the following primary antibodies: anti-human αSMA (dilution 1:300, Sigma-Aldrich), S100A4 (dilution 1:100, Santa Cruz Biotechnology, Dallas, USA), COL1 (dilution 1:600, Proteintech), and FN (dilution 1:2,000, Sigma-Aldrich). The membranes were subsequently incubated with (HRP)-conjugated secondary antibodies (dilution 1:2,000; Cell Signaling, MA, USA). To confirm similar loading of protein samples into the gels and the efficiency in the electrophoretic transfer, membranes were incubated with primary HRP-conjugated antibody to human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:2,000; Santa Cruz Biotechnology). Protein synthesis was detected using the enhanced chemiluminescence system (Luminata Crescendo, Millipore), and the densitometric analysis was performed by the UVITEC Image Analysis System (UVITEC, Cambridge, UK).
3
Western Blot Analysis of Protein Phosphorylation
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Cells were lysed in lysis buffer (20 mmol/L Tris HCl [pH 7.5], 150 mmol/L NaCl, 1 mmol/L Na2‐EDTA, 1 mmol/L EGTA, 1% NP40, 2.5 mmol/L Na2P2O7, and 1 mmol/L β‐glycerophosphate). One mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 1 mmol/L NaF, and a protease inhibitor cocktail (Thermo Fisher Scientific) were added to the buffer immediately before lysing.
The following primary antibodies were used (clones are indicated for monoclonal antibodies): anti–phosphorylated AKT (Ser473, clone D9E; Cell Signaling Technology), anti–phosphorylated AKT (Thr308), anti‐AKT (H‐136), anti–phosphorylated NOS‐3 (Ser1177, 15E2; Santa Cruz Biotechnology), anti–NOS‐3 (6H2; Cell Signaling Technology), anti‐TRF2 (4S794.15; Imgenex, Novus Biologicals), anti–phosphorylated p53 (Ser15, D4S1H; Cell Signaling Technology), anti‐p53, anti‐GAPDH (FL‐335), anti‐AR (Millipore), and anti‐ERK1/2. After incubation with proper horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology), bands were visualized with Clarity Western ECL Substrate (Bio‐Rad) and quantified by densitometry using an image analysis system (UVItec). The quantity of phosphorylated AKT, NOS‐3, and p53 was normalized for the amount of total protein. Levels of GAPDH and ERK1/2, respectively, were used to normalize the expression of TRF2 and p53 at 24 and 48 hours and the expression of AR at 7, 14, and 21 days.
The following primary antibodies were used (clones are indicated for monoclonal antibodies): anti–phosphorylated AKT (Ser473, clone D9E; Cell Signaling Technology), anti–phosphorylated AKT (Thr308), anti‐AKT (H‐136), anti–phosphorylated NOS‐3 (Ser1177, 15E2; Santa Cruz Biotechnology), anti–NOS‐3 (6H2; Cell Signaling Technology), anti‐TRF2 (4S794.15; Imgenex, Novus Biologicals), anti–phosphorylated p53 (Ser15, D4S1H; Cell Signaling Technology), anti‐p53, anti‐GAPDH (FL‐335), anti‐AR (Millipore), and anti‐ERK1/2. After incubation with proper horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology), bands were visualized with Clarity Western ECL Substrate (Bio‐Rad) and quantified by densitometry using an image analysis system (UVItec). The quantity of phosphorylated AKT, NOS‐3, and p53 was normalized for the amount of total protein. Levels of GAPDH and ERK1/2, respectively, were used to normalize the expression of TRF2 and p53 at 24 and 48 hours and the expression of AR at 7, 14, and 21 days.
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