Z. mobilis His-ZMO0038 and His-ZMO0038M strains were cultured in RMG2 medium. Cells were then harvested by centrifugation when OD600 nm reached 0.4, followed by suspension in 50 mM phosphate buffer for sonication lysis. The crude protein samples were fractionated on 12% SDS-PAGE and transferred onto a nylon membrane using the Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad, Hercules, CA, USA). The membrane was incubated with a hybridization buffer including an antibody against His-tag peptide (GenScript, Nanjing, China). The antibody bound to the His-tagged proteins was then recognized by a secondary antibody (Goat Anti-Mouse IgG, GenScript, Nanjing, China). The results were then visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and analyzed using the Amersham Imager 600 device (GE Healthcare, Seoul, South Korea).
Amersham imager 600 device
Manufactured by GE Healthcare
Sourced in United States
The Amersham Imager 600 is a versatile imaging device designed for the analysis of gels, membranes, and other biological samples. It utilizes a high-resolution camera, advanced optics, and specialized software to capture and analyze images of a wide range of samples, including Western blots, DNA gels, and protein gels.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by GenScript (1 mentions)
Semi dry electrophoretic transfer cell system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Secondary goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Semi dry transfer device, by Bio-Rad (1 mentions)
Goat anti mouse hrp secondary antibody, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
P jnk, by Abcam (1 mentions)
Beclin 1, by Abcam (1 mentions)
3 protocols using amersham imager 600 device
1
Western Blot Analysis of His-Tagged Proteins
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Z. mobilis His-ZMO0038 and His-ZMO0038M strains were cultured in RMG2 medium. Cells were then harvested by centrifugation when OD600 nm reached 0.4, followed by suspension in 50 mM phosphate buffer for sonication lysis. The crude protein samples were fractionated on 12% SDS-PAGE and transferred onto a nylon membrane using the Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad, Hercules, CA, USA). The membrane was incubated with a hybridization buffer including an antibody against His-tag peptide (GenScript, Nanjing, China). The antibody bound to the His-tagged proteins was then recognized by a secondary antibody (Goat Anti-Mouse IgG, GenScript, Nanjing, China). The results were then visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and analyzed using the Amersham Imager 600 device (GE Healthcare, Seoul, South Korea).
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Z. mobilis His-ZMO0038 and His-ZMO0038M strains were cultured in RMG2 medium. Cells were then harvested by centrifugation when OD600 nm reached 0.4, followed by suspension in 50 mM phosphate buffer for sonication lysis. The crude protein samples were fractionated on 12% SDS-PAGE and transferred onto a nylon membrane using the Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad, Hercules, CA, USA). The membrane was incubated with a hybridization buffer including an antibody against His-tag peptide (GenScript, Nanjing, China). The antibody bound to the His-tagged proteins was then recognized by a secondary antibody (Goat Anti-Mouse IgG, GenScript, Nanjing, China). The results were then visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and analyzed using the Amersham Imager 600 device (GE Healthcare, Seoul, South Korea).
2
Cry1Ab Toxin Oligomerization Assay
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Cry1Ab oligomers were produced by incubation of 1 μg of purified toxin with 10 μg of BBMV protein in 1 M of buffer NaHCO3 with a pH of 10.5 during 1 h at 30 °C. After incubation of Cry1Ab toxin with BBMVs, samples were ultra-centrifuged for 30 min at 55,000 rpm, 4 °C and the membrane pellet was recovered for further analysis. Oligomerization reactions were performed in presence or absence of reducing agents (0.02% 2-ME or 5 mM DTT). The samples were suspended in Laemmli loading buffer without 2-ME, heated at 50 °C for 3 min, separated by SDS-PAGE and detected by western blot using polyclonal anti-Cry1Ab and secondary goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA). Finally, oligomers were visualized with Luminol reagent (Santa Cruz Biotechnology, Dallas, TX, USA) in Amersham Imager 600 device (GE LifeScience, Little Chalfont, UK).
3
Western Blot Analysis of Signaling Proteins
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Cell lysates were prepared, electrophoresed on 12% SDS-polyacrylamide gels, and then transferred to PVDF membranes using a Semi-dry transfer device (Bio-Rad, USA). Immunoreactive proteins were detected with antibodies to p-JNK (Abcam, Cambridge, UK) or BECLin-1 (Abcam, Cambridge, UK) using ECL (Thermo Scientific, Waltham, MA, USA) and a goat anti-mouse HRP secondary antibody (Abcam, Cambridge, UK). Blots were exposed to film for 1–2 h in the Amersham Imager 600 device (GE healthcare, Pittsburgh, PA, USA).
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