A 1:100 dilution in fresh LB medium, plus appropriate antibiotics, of the overnight cultures used in the spontaneous reversion of hisG4(oc) assay (see above) were incubated at 37 °C until they reached early exponential phase. A 1.0 ml aliquot of each culture was centrifuged, and the resulting cell pellet was suspended in 4 x SDS sample buffer [40% glycerol (v/v), 9.2% SDS (w/v), 4 mM DTT, 250 mM Tris–HCl (pH 6.8), and 0.2% (w/v) bromophenol blue]. Equal protein concentrations of the aliquots were determined using the Pierce BCA Protein Assay (ThermoFisher Scientific) and electrophoresed on 12% SDS-polyacrylamide gels. Next, proteins were transferred to an Immobilon P membrane (Millipore) and subsequently incubated with polyclonal antibodies directed against the RumA protein (Covance). Visualization of the transferred RumA protein was performed on Kodak BioMaxMR film using the BioRad Immun-Star AP substrate.
Biomaxmr film
Manufactured by Bio-Rad
Sourced in United States
BioMaxMR film is a laboratory equipment product designed for high-resolution imaging of radioisotope-labeled samples. It is a versatile film that can be used for various applications, such as autoradiography, blotting, and quantitative analysis of radioactive samples. The film provides excellent sensitivity and resolution, making it suitable for a wide range of research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (1 mentions)
Immun star ap substrate, by Bio-Rad (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Gel dryer 583, by Bio-Rad (1 mentions)
Whatman filter paper, by GE Healthcare (1 mentions)
Fuji fla5000 phosphorimager, by Fujifilm (1 mentions)
2 protocols using biomaxmr film
1
Western Blot Analysis of RumA Protein
2
Native PAGE Gel Visualization of 35S-Labeled Proteins
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Native PAGE gels were incubated in 100% v/v DMSO for 20 min at room temperature on an orbital shaker. The gel was incubated in fresh 100% (v/v) DMSO for 20 min, followed by incubation in 22% (w/v) 2,5-diphenyloxazole in DMSO overnight. The gels were washed multiple times in fresh, deionized water until all precipitated residue had been removed. The gels were dried onto Whatman filter paper (GE Healthcare, Little Chalfont, United Kingdom) using a Bio-Rad Gel Dryer 583. 35S-Labeled proteins were visualized by autoradiography using BioMax MR film (Bio-Rad, Hercules, CA, USA) or a Fuji FLA-5000 PhosphorImager (Fujifilm, Tokyo, Japan). Band intensities were quantified using AIDA software (FinalWire, Budapest, Hungary), and images were analyzed and processed using ImageJ (U.S. National Institues of Health, Bethesda, MD, USA).
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