Proteins were electroblotted onto a low-fluorescent PVDF membrane using a Trans-Blot Turbo transfer cell and the RTA transfer kit (Bio-Rad Laboratories) for 10 min (25 V, 1.3 A, RT) and processed as described [31 (link)]. For detection, the membrane was incubated with Immuno Blue HRP-Substrate (NH DyeAGNOSTICS, Halle, Germany) for 10 min or with WesternBright Sirius HRP-Substrate (Advansta, Menlo Park, CA, USA) for 2 min, washed in Advan washing solution, and the fluorescence or chemoluminescence recorded (ChemiDoc MP CCD camera system, Bio-Rad Laboratories).
Chemidoc mp ccd camera system
Manufactured by Bio-Rad
Sourced in United States, Germany
The ChemiDoc MP CCD camera system is a high-performance imaging device designed for a variety of laboratory applications. It utilizes a charge-coupled device (CCD) sensor to capture and digitize images of chemiluminescent, fluorescent, and colorimetric samples. The system is capable of generating high-quality, high-resolution images for tasks such as protein, nucleic acid, and gel documentation.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer cell, by Bio-Rad (2 mentions)
Westernbright sirius hrp substrate, by Advansta (1 mentions)
Rta transfer kit, by Bio-Rad (1 mentions)
Mini protean 3 cell, by Bio-Rad (1 mentions)
Powerpac 300, by Bio-Rad (1 mentions)
Trans blot turbo rta transfer kit, by Bio-Rad (1 mentions)
4 protocols using chemidoc mp ccd camera system
1
Protein Electroblotting and Detection
2
Protein Immunoblotting Assay Protocol
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Proteins were electroblotted onto a PVDF membrane using a Trans-Blot Turbo transfer cell (Bio-Rad Laboratories) for 10 min (25 V, 1.3 A, RT). Membranes were blocked with Advan Blocking solution (30 min, RT) and incubated with serum samples (3 μL serum in 10 mL Advan Blocking solution) overnight at 4 °C. Afterwards, membranes were washed two times with Advan Washing solution (5 min), incubated with peroxidase-conjugated goat anti-mouse IgG + IgM (0.5 μL in 10 mL Advan blocking solution plus 0.5% BSA (w/v), 90 min, RT), and washed again with Advan Washing solution (3×, 5 min each). The membrane was incubated with Immuno Blue HRP-Substrate (NH Dyeagnostics, Halle, Germany) for 10 min, washed in Advan washing solution and the fluorescence was recorded (ChemiDoc MP CCD camera system, Bio-Rad Laboratories).
3
SDS-PAGE Analysis of Protein Samples
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Protein samples and fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a Mini-Protean 3 cell (Bio-Rad Laboratories) using stacking (T = 4%, C = 2.67%) and separation gels (T = 12%, C = 2.67%; 6 cm long) that were 1 mm thick. Samples were diluted (1:4, v/v) with sample buffer (62.5 mmol/L Tris/HCl, pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 0.5% (w/v) bromophenol blue) to a total volume of 20 μL, heated (5 min, 95 °C), and loaded on the stacking gel. Separation was performed by applying voltages of 100 V for 10 min and 200 V for 45 min using a PowerPac 300 (Bio-Rad Laboratories). Proteins were visualized by colloidal Coomassie Brilliant Blue (CBB) G-250 [32 (link)], or Oriole™ fluorescent stain (λexc = 270 nm, λem = 604 nm) according to the manufactures protocol. Images were taken on a ChemiDoc MP CCD camera system (Bio-Rad Laboratories).
4
Protein Separation and Immunodetection
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Proteins were mixed with 5x sample buffer (0.5% bromophenol blue, 62.5 mmol/L Tris-HCl, pH 6.8, 20% glycerol, 2% sodium dodecyl sulfate (SDS), 5% ß-mercaptoethanol), incubated at 95 °C for 10 min, and separated by SDS-PAGE (T = 16%, C = 2.67 %). Gels were stained with Oriole (Oriole Fluorescent Gel Stain; Bio-Rad Laboratories, Feldkirchen, Germany) for 60 min and Coomassie overnight or blotted onto a LF-PVDF membrane using the TransBlot Turbo RTA Transfer Kit (Bio-Rad Laboratories) at 2.5 V for 7 min. The membrane was incubated with the particle-based immunoassay PIA-Pink technology, which includes washing with PIA-Pink-Block for 5 min and incubation overnight (4 °C, 30 rpm) with PIA-Pink-Mouse (PiNa-Tec, Hamburg, Germany) containing 2 µg of anti-biotin mouse mAb (Life Technologies GmbH, Darmstadt, Germany). The membrane was dried for one hour at room temperature and photographed (ChemiDoc MP CCD camera system, Bio-Rad Laboratories).
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