We extracted the bacterial protein with NP40 lysis buffer (50 mM Tris, pH 8.5, 150 mM NaCl, 1% NP40) with protease inhibitor cocktail VII (Research Product International, Mt. Prospect, IL, USA) and performed SDS-PAGE with 50 μg/lane of proteins in 7.5 or 12% acrylamide gel. Then, some gels were stained directly with GelCodeBlue Stain Reagent (Thermo Fisher) and others were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) using Mini Trans-Blot Cell and Criterion Blotter (Bio-Rad). Efficiency of the transfer was confirmed by a brief staining of the membranes with Ponceau S solution. Expression levels of the proteins in bacteria were evaluated by western blotting with specific antibodies. Primary antibodies used in the immunoblotting are summarized in Table 1 . Secondary antibody (HRP-conjugated anti mouse IgG) was obtained from Cell Signaling Technologies (#7076, Danvers, MA, USA) and used in 2000 times dilution. Western blots with chemiluminescence (SuperSignal West Pico PLUS; Thermo Fisher) were developed by ChemiDoc Imager (Bio-Rad) and then analyzed using Image Lab software (Bio Rad).
Mini trans blot cell and criterion blotter
Manufactured by Bio-Rad
Sourced in United States
The Mini Trans-Blot Cell is a compact, self-contained electrophoretic transfer system designed for protein blotting. The Criterion Blotter is a semi-dry blotting system for transferring proteins from polyacrylamide gels to membranes.
Automatically generated - may contain errors
Lab products found in correlation
Gelcode blue stain reagent, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Tween 20, by Merck Group (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
Protease inhibitor, by G Biosciences (1 mentions)
Bovine serum albumin (bsa), by MP Biomedicals (1 mentions)
β mercaptoethanol, by Bioworld Technology (1 mentions)
3 protocols using mini trans blot cell and criterion blotter
1
Bacterial Protein Extraction and Analysis
2
Quantifying CD31 Protein Levels in Mouse Ovaries
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Protein samples obtained from mouse ovaries were incubated with PRO-PREPTM Protein Extraction Solution (iNtRON Biotechnology, Seongnam, South Korea). Twenty micrograms of proteins were separated using 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane using a Mini Trans-Blot cell and criterion blotter (Bio-rad, Hercules, CA, USA). After blocking with 5% skim milk in 1× TBST with 0.1% Tween 20 (Sigma-Aldrich) at room temperature for 1 hour, the blots were probed at 4°C overnight with a 1:300 dilution of anti-CD31 primary antibodies (BS-0195R; Bioss, Woburn, MA, USA) and a 1:1000 dilution of anti-β-tubulin (s2128; Cell Signaling Technology, Danvers, MA, USA). This was followed by incubation in a 1:2000 dilution of mouse anti-rabbit IgG secondary antibody (sc-2357; Santa Cruz Biotechnology, Dallas, TX, USA) at room temperature for 1 hour. Band intensity was quantified using Image J and normalized to β-tubulin band intensity.
3
Western Blot Protein Quantification Protocol
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Protein levels were analyzed using western blot. Briefly, cells were harvested and washed with 1× phosphate buffered saline and lysed in a lysis buffer (RIPA buffer), Na-vanadate (1 mM), β-glycerol phosphate (50 mM), protease inhibitor (cat#786-108, G-biosciences, St. Louis, MO), EDTA (5 mM), β-mercaptoethanol (cat#41300000-1, 142 mM, BioWORLD, Dublin, OH). The cell lysate was mixed with 5× sample buffer and the mixture was boiled at 100°C for 10 min. Subsequently, the samples were loaded on 12% and 15% polyacrylamide gels. Proteins were transferred to membranes using Mini Trans-Blot Cell and Criterion Blotter (Bio-Rad, Hercules, CA) and the membranes were blocked in 1% BSA (bovine serum albumin, cat#160069, MP Biomedicals, Santa Ana, CA) dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST). The membranes were probed by indicated primary antibodies and incubated on a rotator at 4°C overnight. Then, the membranes were washed for 5 minutes in TBST and treated with anti-mouse or -rabbit secondary antibodies for 1 hour at room temperature. After washing three times for 10 minutes each with TBST, proteins were detected using a chemiluminescent substrate (EZ west Lumi Plus, ATTO, Tokyo, Japan) and visualized on a chemiluminescence Imaging system (Luminograph II, ATTO).
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