Saliva samples (20 μg) were electrophoretically separated on 10–20% Tris-glycine gels (1.5 mm thick, Novex by Life Technologies) and transferred to nitrocellulose membranes, followed by blocking for 1 h with 5% nonfat powdered milk in PBS-Tween before incubating overnight at 4 °C with the primary antibodies. Primary antibodies used were anti-BPIFA2 (Novus Biologicals), anti-μ2 M (Santa Cruz Biotechnology), and anti-CAH6 (Abcam). Then, the membranes were washed three times in PBS-Tween and incubated with a secondary antibody (Peroxidase AffiPure Donkey Anti-Rabbit IgG or Peroxidase AffiPure Donkey Anti-Mouse IgG from Jackson ImmunoResearch) at a 1:5000 dilution for 1 h. Protein bands were detected with ECL 2 Western Blotting Substrate (Thermo Scientific, Pierce) and ECL high-performance chemiluminescence film (Amersham, GE Healthcare).
Ecl high performance chemiluminescence film
Manufactured by GE Healthcare
ECL high-performance chemiluminescence film is a laboratory equipment product designed for capturing and recording chemiluminescent signals generated during Western blotting experiments. It provides high sensitivity and resolution for the detection of low-abundance proteins.
Automatically generated - may contain errors
2 protocols using ecl high performance chemiluminescence film
1
Western Blot Analysis of Salivary Proteins
2
Western Blot Analysis of CTB Cell Proteins
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Proteins from isolated CTB cell pellets were solubilized in M-PER (Thermo Fisher Scientific) according to the manufacturer's protocol and protein concentrations determined spectrophotometrically (Shimadzu). Samples (5 μg) were electrophoretically separated on 4-12% Bis-Tris protein gels (1.5 mm, Invitrogen) and transferred to nitrocellulose membranes, followed by blocking for 1 h with 5% nonfat powdered milk in PBS-Tween-20 (0.1%) before incubating overnight at 4°C with primary antibody. Then the membranes were washed three times in PBS-Tween-20 and incubated with the appropriate species-specific secondary antibody for 1 h. Immunoreactive bands were detected with ECL 2 Western Blotting Substrate (Thermo Fisher Scientific, Pierce) and ECL high performance chemiluminescence film (Amersham, GE Healthcare). Equal sample loading in all the lanes was confirmed by anti-α-actin immunoreactivity.
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