Gonococcal outer membrane vesicles (OMV) were prepared by shearing in lithium acetate buffer as described previously (35 (link)). OMV preparations were boiled for 5 min in SDS loading buffer containing 2-mercaptoethanol. Protein quantification was done with the RC DC protein assay kit (Bio-Rad, Hercules, CA). From each sample, 10 μg of protein was electrophoresed on 10% Mini-PROTEAN TGX precast gels (Bio-Rad). A prestained protein ladder (PageRuler; Thermo Fisher Scientific) was used as a molecular mass marker. Replicate gels were either stained with Coomassie blue or transferred to nitrocellulose membranes using the electrophoresis transfer system (Bio-Rad). Membranes were blocked in PBS containing 3% skim milk overnight at 4°C and incubated for 2 h with serum samples diluted 1:200 in PBS plus 3% skim milk. Specific antibodies bound to N. gonorrhoeae OMV preparations were detected with horseradish peroxidase-conjugated goat anti-mouse IgG or anti-mouse IgA (Santa Cruz Biotechnology, Paso Robles, CA) diluted 1:4,000 in PBS plus 3% skim milk. The Pierce (Rockford, IL) detection kit was used for chemiluminescence detection, and images were collected with a ChemiDoc MP imaging system (Bio-Rad).
Electrophoresis transfer system
Manufactured by Bio-Rad
Sourced in United States
The Electrophoresis Transfer System is a laboratory equipment used to transfer proteins or nucleic acids from a gel to a membrane for further analysis. It provides a controlled and efficient method for the transfer process, allowing for the detection and study of the transferred biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Santa Cruz Biotechnology (3 mentions)
Pageruler, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Rc dc protein assay kit, by Bio-Rad (1 mentions)
Anti his antibody, by Qiagen (1 mentions)
Piercetm, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Respective secondary antibodies, by Abcam (1 mentions)
Imagej software, by Bio-Rad (1 mentions)
Enhanced chemiluminescence reaction, by Thermo Fisher Scientific (1 mentions)
7 protocols using electrophoresis transfer system
1
Gonococcal Outer Membrane Vesicle Characterization
2
Extraction and Detection of scFvs
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The scFvs localized in periplasmic space were extracted using BugBusterTM and 10 µL of total proteins was analyzed by SDS-PAGE via a standard method using 9% polyacrylamide ready-to-use gels (Anamed, Groß-Bieberau, Germany). Gels were either stained with Coomassie blue staining reagent (Fermentas, ThermoFisher Scientific) or were transferred to polyvinylidenfluorid (PVDF) membranes using an electrophoresis transfer system (Bio-Rad). ScFvs were detected using anti-His antibody (Qiagen, Hilden, Germany) followed by anti-mouse conjugated peroxidase (POD). Bound antibodies were detected by enhanced chemiluminescence reaction (PierceTM, ThermoFisher Scientific).
3
Immunoblotting of N. gonorrhoeae OMVs
4
Immunoblotting of N. gonorrhoeae OMVs
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N. gonorrhoeae OMV preparations were boiled for 5 min in sodium dodecyl sulfate (SDS) loading buffer containing 2-mercaptoethanol. Protein quantification was done with the RC DC Protein Assay kit. Ten micrograms of protein from each sample was separated on 10% polyacrylamide SDS electrophoresis gels. Protein bands were transferred onto nitrocellulose membranes using the electrophoresis transfer system (Bio-Rad, Hercules, CA, USA). The nitrocellulose membranes were blocked with PBS containing 3% skim milk overnight at 4°C before incubation for 2 h with serum samples diluted 1:200, or vaginal wash samples diluted 1:20 in PBS containing 3% skim milk. Specific antibodies bound to N. gonorrhoeae OMV preparations were detected with horseradish peroxidase-conjugated goat anti-mouse-IgG (Santa Cruz Biotechnology, Paso Robles, CA) at a dilution of 1:4000. The Pierce detection kit was used for chemiluminescent detection and images were collected with a ChemiDoc MP imaging system (Bio-Rad).
5
Western Blot Analysis of PTER in Hepatoma Cells
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Western blotting was carried out as described previously (7 (link)). Briefly, SMMC-7721 hepatoma cells were treated with various concentrations (5, 25, 50, or 100 µM) of PTER for 24 h. PTER (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulphoxide (DMSO); DMSO alone was used to treat cells in the control group. After washing with PBS, cells were then lysed using RIPA buffer, which was mixed with the Halt Phosphatase and Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc., Waltham, MA, USA). After the protein concentration was quantified, 70 µg of protein was resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane using an Electrophoresis Transfer System (Bio-Rad). Subsequently, membranes placed on a swing table were blocked with TBS-Tween and 5% dry milk and were then probed with anti-MTA1, p53 or Ac-p53 (Abcam, Cambridge, UK) primary antibodies overnight at 4°C. Respective secondary antibodies (Abcam) were used at 1:1,000–1:2,000 (v/v) dilutions in TBS-Tween and 5% fat-free powdered milk for 1 h at room temperature; β-actin antibodies were used as a loading control. Finally, blots were visualized by SuperSignal West Pico enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the density of the blots was quantified using Image J Software (Bio-Rad).
6
Fusion Protein SDS-PAGE Analysis
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Fusion proteins were analyzed by SDS-PAGE standard methods using 9% polyacrylamide ready-to-use gels (anamed Elektrophorese GmbH, Groß-Bieberau, Germany). Gels were either stained with coomassie blue staining reagent or transferred to PVDF membranes using an electrophoresis transfer system (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Imp-PM19 and Imp-PM19 mutant fusion proteins were detected using monoclonal anti-His antibodies (Quiagen, Hilden, Germany) specific for the His-tag, following anti-Mouse-POD. Bound antibodies were detected by an enhanced chemiluminescence reaction (Thermo Fisher Scientific, Karlsruhe, Germany).
7
Western Blot Analysis of Gonococcal OMVs
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Gonococcal OMV preparations were boiled for 5 min in SDS loading buffer containing 2-mercaptoethanol. Protein quantification was done with the RC DC protein assay kit. Twenty micrograms of protein from each sample was separated on 10% polyacrylamide–SDS electrophoresis gels. Protein bands were transferred onto nitrocellulose membranes using the electrophoresis transfer system (Bio-Rad, Hercules, CA, USA). The membranes were blocked with PBS containing 3% skim milk overnight at 4°C before incubation for 2 h with serum samples diluted 1:200 or vaginal wash samples diluted 1:20 in PBS containing 3% skim milk. Specific antibodies bound to N. gonorrhoeae OMV preparations were detected with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Paso Robles, CA) at a dilution of 1:4,000. The Pierce detection kit was used for chemiluminescent detection, and images were collected with a ChemiDoc MP imaging system (Bio-Rad). Replicate gels were stained using Coomassie blue to reveal protein bands.
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