Protein expression was analyzed in both cell lysates and cell supernatants as previousely described [34 (link)]. Briefly, after debris removal, cell culture supernatants were precipitated with ice-cold acetone and cell lysates were prepared in Pierce IP Lysis Buffer, containing Halt Protease and Phosphatase Inhibitor (Thermo Fisher Scientific, Boston, MA, USA). Protein concentrations were determined with the Bradford quantification assay (Bio-Rad Laboratories, Des Plaines, IL, USA), using BSA (Sigma-Aldrich; St. Louis, MO, USA) as a standard. Equal protein amounts of cell lysates or equal volumes of cell supernatants were prepared in NuPAGE LDS Sample Buffer (Thermo Fisher Scientific, Boston, MA, USA), containing reducing agent (Thermo Fisher Scientific, Boston, MA, USA) and analyzed on Novex 8–16% Tris-Glycine gels (Invitrogen, Waltham, MA, USA). Subsequently, proteins were transferred onto PVDF membranes (Roche, Basel, Switzerland), and Western blot detection was carried out as previously described [33 (link),34 (link)], using the primary antibodies listed in Table 2 . Pictures were acquired with a FusionFX imaging system (Vilber Lourmat, Collégien, France), and the intensity density of individual bands was quantified using ImageJ (Version 1.53a).
Bradford quantification assay
Manufactured by Bio-Rad
Sourced in United States
The Bradford quantification assay is a colorimetric method for the determination of protein concentration. It utilizes the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Reducing agent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Fusion fx imaging system, by Vilber (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Novex 8 16 tris glycine gels, by Thermo Fisher Scientific (1 mentions)
3 protocols using bradford quantification assay
1
Protein Expression Analysis in Cell Lysates and Supernatants
2
Quantifying Quorum Sensing Molecules
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Concentrations of HAQs and AHLs in cultures were measured for bacteria grown in King's A medium. Based on their production profile, concentrations of 3-oxo-C 12 -HSL, C 4 -HSL were measured at 3 h (1) and 6 h (2) time points. The main C 7 HAQ congeners HHQ, HQNO and PQS were measured at 6 h (1) and 24 h (2). Briefly, an equal volume of methanol containing internal standard tetradeuterated 4-hydroxy-2-heptylquinoline (HHQ-d4) was added to the culture sample. The suspension was vortexed and centrifuged for 5 min at maximum speed to remove bacteria. The resulting supernatant was transferred in vials for liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analyses, as previously reported (Lépine et al., 2018) For quantifications of AHLs, ethyl acetate extracts were analysed by LC/MS/MS as described (Lépine et al., 2018) . The cell pellets were suspended in 0.1 N NaOH and heated at 70 C for 1 h with frequent vortexing. Protein concentrations were obtained using the Bradford quantification assay (Bio-Rad). Production (mg l À1 ) is presented as ratios over biomass (μg ml À1 of proteins).
3
Quantifying Quorum Sensing Signals
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Concentrations of HAQs and AHLs in cultures were measured for bacteria grown in King's A medium. Based on their production profile, concentrations of 3-oxo-C 12 -HSL, C 4 -HSL were measured at 3h (1) and 6h (2) time points. The main C 7 HAQ congeners HHQ, HQNO and PQS were measured at 6h (1) and 24h (2) . Briefly, an equal volume of methanol containing internal standard tetradeuterated 4-hydroxy-2-heptylquinoline (HHQ-d4) was added to the culture sample. The suspension was vortexed and centrifuged 5 min at maximum speed to remove bacteria. The resulting supernatant was transferred in vials for liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analyses, as previously reported (45) For quantifications of AHLs, ethyl acetate extracts were analyzed by LC/MS/MS as described (45) .
The cell pellets were suspended in 0.1N NaOH and heated at 70°C for 1 hour with frequent vortexing. Protein concentrations were obtained using the Bradford quantification assay (BioRad). Production (mg/L) is presented as a ratios over biomass (µg/mL of proteins).
The cell pellets were suspended in 0.1N NaOH and heated at 70°C for 1 hour with frequent vortexing. Protein concentrations were obtained using the Bradford quantification assay (BioRad). Production (mg/L) is presented as a ratios over biomass (µg/mL of proteins).
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