Total proteins were extracted using Pierce cell lysis buffer (Pierce, Thermo Fisher Scientific, Inc., Rockford, IL, USA). The proteins were quantified by the Bradford method. A total of 50 µg of protein was transferred onto PVDF membranes following separation by SDS-PAGE. The membranes were incubated at 4°C overnight with primary antibodies against Eya2 (1:1,000; 11314-1-AP; Proteintech), Six1 (1:1,000; SAB2104425; Sigma-Aldrich), cyclin D1 (2978), cyclin E (20808), p-ERK (9101), ERK (9102), MMP9 (3852) and GAPDH (2118) (1:1,000; all from Cell Signaling Technology, Inc., Boston, MA, USA). Following incubation with HRP-coupled anti-mouse (sc-2789)/rabbit (sc-2357) IgG (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 37°C for 2 h, the bound proteins were visualized using an ECL kit (Pierce) and detected using a DNR Bio-Imaging System (DNR Bio-Imaging Systems, Ltd., Jerusalem, Israel). The densitometry of western bands was measured using ImageJ software.
Dnr bio imaging system
Manufactured by DNR Bio-Imaging Systems
Sourced in Israel
The DNR Bio-Imaging System is a versatile laboratory equipment designed for researchers and scientists. It provides high-quality image capture and analysis capabilities for a variety of biological samples and applications.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (3 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Gapdh 2118, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
P iκb, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Peroxidase coupled anti mouse rabbit igg, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Glut1, by Proteintech (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
6 protocols using dnr bio imaging system
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Signaling Proteins
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Total proteins from HeLa and CaSki cells were extracted and quantified using the Bradford method, and 20 mg protein was separated by SDS-PAGE. Samples were transferred to polyvinylidene difluoride membranes (EMD Millipore) and incubated overnight at 4°C with antibodies against CrkL (rabbit polyclonal; dilution, 1:1,000; cat. no. ABC242; EMD Millipore), p-Src (rabbit monoclonal; dilution, 1:1,000; cat. no. 12432; Cell Signaling Technology, Inc., Boston, MA, USA), Src (rabbit monoclonal; dilution, 1:1,000; cat. no. 2109; Cell Signaling Technology, Inc.), p-Akt (rabbit polyclonal; dilution, 1:1,000; cat. no. 9271; Cell Signaling Technology, Inc.), Akt (rabbit polyclonal; dilution, 1:1,000; cat. no. 9272; Cell Signaling Technology, Inc.; dilution, 1:1,000) and GAPDH (rabbit polyclonal; dilution, 1:1,000; cat. no. G5262; Santa Cruz Biotechnology, Inc.). Following incubation with peroxidase-coupled anti-mouse/rabbit IgG (dilution, 1:2,000; cat. no. 5127; Cell Signaling Technology, Inc.) at 37°C for 2 h, proteins were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and detected using a DNR Bio-Imaging System (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).
3
Quantification of Apoptosis Proteins
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Total protein of MCF-7 cells was extracted using lysis buffer (Pierce Biotechnology, Inc., Rockford, IL, USA) and Bradford method was used to quantify the protein. When SDS-PAGE assay was performed, 30 µg of the protein was separated and then transferred to polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). For primary antibody incubation, cleaved caspase 3, caspase 3, cleaved PARP, PARP, p-p65, p65, p-IκB, IκB, Bcl-2 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), and GAPDH (1:2,000; Cell Signaling Technology, Inc.) were incubated overnight at 4°C. For secondary antibody incubation, peroxidase-coupled anti-mouse/rabbit IgG (1:1,000; Cell Signaling Technology, Inc.) was incubated at 37°C for 2 h. Sample protein was visualized using ECL (Pierce Biotechnology, Inc.) and detected using a DNR BioImaging System (DNR Bio-Imaging Systems, Ltd., Jerusalem, Israel). Relative protein levels were quantified using ImageJ software.
4
Western Blot Analysis of CIP2A, c-Myc, and PARP
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Cell lysates were prepared using lysis buffer with protease and phosphatase inhibitor. Total protein was extracted and quantified. Sample protein (40 μg) was resolved through SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Then PVDF membranes were blocked with 5% BSA for 1 hour at room temperature. Primary antibodies CIP2A c-Myc, cleaved PARP, and actin (1:1000, Cell Signal Technology, USA) were incubated at 4°C overnight. PVDF membranes were rinsed with TTBS buffer and incubated with secondary anti-mouse/rabbit IgG HRP-linked antibody (1:2000, Cell Signal Technology, USA) at 37°C for 1 hour. Proteins were visualized by ECL kit (Thermo Fisher Scientific; USA) in a DNR Bio-Imaging system (DNR Bio-Imaging systems, Israel).
5
Western Blot Analysis of Metabolic Proteins
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Total protein was extracted using RIPA lysis and extraction buffer (Thermo Fisher Scientific, Inc.) and quantified via the Bradford method. Proteins (50 µg/lane) were separated via 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% milk at room temperature for 1 h and then incubated with antibodies against BCAT1 (1:1,500; cat. no. 13640-1-AP; ProteinTech Group, Inc.), GLUT1 (1:1,000; cat. no. 12939; Cell Signaling Technology, Inc.), c-Myc (1:1,000; cat. no. 5605; Cell Signaling Technology, Inc.), and GAPDH (1:3,000; cat. no. 5174; Cell Signaling Technology, Inc.). After incubation with HRP-coupled secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.), protein bands were visualized using a Pierce™ Fast Western Blot kit ECL Substrate (Thermo Fisher Scientific, Inc.). Images were captured using a DNR bio-imaging system (DNR bio-imaging systems, Ltd.).
6
Western Blot Analysis of DEF6 Protein
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Total proteins from cells were extracted using the NP-40 lysis buffer (cat. no. P0013F; Beyotime Institute of Biotechnology) and quantified using the BCA method. The supernatant (20 µg of protein) was denatured and separated on 10% SDS-PAGE. Samples were then transferred to 0.22-µm PVDF membranes and blocked in skimmed milk for 1 h at room temperature. After that, samples were incubated overnight at 4°C with the antibodies against DEF6 (cat. no. ab247011; 1:1,000; Abcam) and β-actin (cat. no. sc47778; 1:1,000; Santa Cruz Biotechnology, Inc.). After incubation with peroxidase-coupled anti-rabbit IgG (cat. no. 7074; 1:1,000, Cell Signaling Technology, Inc.) at 37°C for 2 h, bound proteins were visualized using an ECL kit (Pierce; Thermo Fisher Scientific, Inc.) and detected using a DNR Bioimaging System (DNR Bio-Imaging Systems, Ltd.). Relative protein levels were quantified using β-actin as the loading control.
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