Confirmation of protein identities was performed by Western blotting. Protein extracts were prepared in lysis buffer and each cell lysate sample (1 μg/μL, 10 μL) was electrophoresed through a precast gel (NuPAGE®Novex® 4–12% Bis-Tris Gel, 1.5 mm, 10 wells, Invitrogen™, Carlsbad, CA). Proteins were transferred from the gel to a polyvinyldifluoride (PVDF) membrane (Millipore, Bedford, CA) by means of the semidry technique using the Criterion Blotter (Bio-Rad) at 100 V for 60 min and blocked with 5% milk in PBS (adjusted to pH 7.4) containing 0.05% Tween-20. The membranes were then separately incubated overnight with primary rabbit antibodies (1 μg/μL). The commercially available primary antibodies used in this study included the following: monoclonal mouse anti-HSP60 (Stressgen, USA) and polyclonal rabbit anti-RanBP2 (Abcam, USA). After washing, the membrane was incubated with HRP-conjugated goat anti-mouse IgG antibodies (purchased from Jackson ImmunoResearch, USA) for 1 hour (1 : 10000). Proteins were detected with an enhanced chemiluminescent (ECL) system, and quantitative analysis of Western blotting was carried out using the ImageQuant-TL-7.0 software, version 2010 (Amersham Biosciences).
Imagequant tl 7.0 software version 2010
Manufactured by Cytiva
Sourced in United States
The ImageQuant-TL-7.0 software, version 2010, is a digital imaging analysis tool used for quantitative analysis of gel-based and blot-based protein and nucleic acid analyses. The software provides image capture, analysis, and data management capabilities for various gel electrophoresis and blotting techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Criterion blotter, by Bio-Rad (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Monoclonal mouse anti hsp60, by Stressgen (1 mentions)
Polyclonal rabbit anti ranbp2, by Abcam (1 mentions)
Polyvinyl difluoride membrane, by Merck Group (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using imagequant tl 7.0 software version 2010
1
Western Blotting Protocol for Protein Identification
2
Protein Extraction and Western Blot Analysis
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Cells were lysed using 2× high-salt radioimmunoprecipitation assay (2× RIPA) buffer and complete protease inhibitor phenylmethylsulfonyl fluoride (PMSF). The concentration of the extracted proteins was determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The proteins were then transferred from SDS-PAGE gel to a polyvinyldifluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% milk in PBS containing 0.05% Tween-20, the membrane was incubated with primary antibodies anti-MAP1S (1:1,000; GeneTex, San Antonio, TX, USA), anti-Ace-tubulin (1:2,000; Sigma-Aldrich), and anti-β-actin (1:1,000; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Later, secondary antibody (1:2,000; Jackson Immunoresearch) was added to it and the membrane was incubated for 60 m at room temperature. Finally, proteins were quantified using the ImageQuant-TL-7.0 software version 2010 (Amersham Biosciences, Piscataway, NJ, USA).
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