G6532

Manufactured by Merck Group

Sourced in United Kingdom

The G6532 is a laboratory equipment product manufactured by Merck Group. It is a general-purpose device used for various laboratory applications. The core function of this product is to perform a specific set of laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

F1804, by Merck Group (2 mentions) Pvdf membrane, by Cytiva (2 mentions) Las4000, by GE Healthcare (1 mentions) Tgx gel, by Bio-Rad (1 mentions) Hybond pvdf membrane, by GE Healthcare (1 mentions) Amersham ecl prime reagent, by GE Healthcare (1 mentions) Laemmli sample buffer, by Bio-Rad (1 mentions) Sab2702197, by Merck Group (1 mentions) Hybond, by Cytiva (1 mentions) Amersham ecl prime, by Cytiva (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Novex ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (1 mentions) Nc membrane, by GE Healthcare (1 mentions) A0545, by Merck Group (1 mentions) Difco skim milk, by BD (1 mentions) A0168, by Merck Group (1 mentions) Image labtm software, by Bio-Rad (1 mentions) D110087, by Sangon (1 mentions) A600669 0250, by Sangon (1 mentions) F1804 5mg, by Merck Group (1 mentions)

6 protocols using g6532

Western Blotting Protocol for Protein Analysis

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Western blotting was performed following a previously published protocol (Miyakoshi et al., 2019 (link)). Briefly, whole‐cell samples in 1× Laemmli sample buffer (Bio‐Rad) were separated on 10% or 12% TGX gels (Bio‐Rad). Proteins were transferred onto Hybond PVDF membranes (GE Healthcare), and membranes were blocked for 10 min in Bullet Blocking One buffer (Nacalai Tesque) and were incubated overnight at 4℃ with monoclonal anti‐FLAG (Sigma–Aldrich #F1804; 1:5,000), monoclonal anti‐GFP (Sigma–Aldrich SAB2702197; 1:5,000), and polyclonal anti‐GroEL (Sigma–Aldrich #G6532; 1:10,000) antibodies diluted in Bullet Blocking One buffer. Membranes were washed three times for 15 min in 1 × TBST buffer at RT. Then membranes were incubated for 1 hr at RT with secondary antimouse or antirabbit HRP‐linked antibodies (Cell Signaling Technology #7076 or #7074; 1:5,000) diluted in Bullet Blocking One buffer and were washed three times for 15 min in 1 × TBST buffer. Chemiluminescent signals were developed using Amersham ECL Prime reagents (GE Healthcare), visualized on LAS4000 (GE Healthcare), and quantified using Image Quant TL software.

Miyakoshi M., Okayama H., Lejars M., Kanda T., Tanaka Y., Itaya K., Okuno M., Itoh T., Iwai N, & Wachi M. (2021). Mining RNA‐seq data reveals the massive regulon of GcvB small RNA and its physiological significance in maintaining amino acid homeostasis in Escherichia coli. Molecular Microbiology, 117(1), 160-178.

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Western Blot for Protein Quantification

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Western blot was performed as described (3 (link),37 (link)). Briefly, 0.1 OD₆₀₀ bacterial culture was collected by centrifugation for 2 min at 8000 rpm at 4°C, and resuspended in 100 μl 1× protein loading buffer. After heating for 5 min at 95°C, 0.02 OD₆₀₀ of cell lysate was separated on a 10% SDS-PAGE gel. Proteins were transferred onto a NC membrane (#10600002, GE Healthcare) for 60 min at 100 V in transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, pH 8.3). Membranes were blocked for 1 h at room temperature in 1× TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween20, pH 7.6) buffer with 5% (w/v) Difco™ skim milk (#6307915, BD). After blocking, membranes were incubated at room temperature for 1 h with monoclonal α-FLAG (Sigma-Aldrich #F1804; 1:1000) or polyclonal α-GroEL (Sigma-Aldrich #G6532; 1:5000) antibodies diluted in 1× TBST buffer containing 3% BSA. Following three washes for 30 min in 1× TBST buffer, membranes were incubated with secondary α-mouse or α-rabbit HRP-linked antibodies (Sigma-Aldrich #A0168 or #A0545; 1:5000) diluted in 1× TBST buffer containing 3% BSA. After another three washes for 30 min in 1× TBST buffer, chemiluminescence was developed using the Novex™ ECL Chemiluminescent Substrate Reagent Kit (#WP20005, Thermo Fisher Scientific), visualized on ChemiDocTM XRS+, and quantified using ImageLabTM Software (both Biorad).

Wang C., Chao Y., Matera G., Gao Q, & Vogel J. (2019). The conserved 3′ UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration. Nucleic Acids Research, 48(4), 2126-2143.

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Fractionation and Characterization of B. thetaiotaomicron Cell Components

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B. thetaiotaomicron strains were grown in minimal medium to log phase. Cells were harvested and separated from the supernatant by centrifugation. Cells were then resuspended in breakage buffer (50 mM Tris [pH 7.4], 5 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 10% glycerol) and lysed by sonication. The lysate and supernatant were filtered (0.2-μm pore size) to remove any whole cells before centrifugation at 100,000 × g at 4°C for 1 h. The pelleted membrane fraction was washed and resuspended in breakage buffer. The soluble and membrane fractions were then centrifuged a second time at 100,000 × g at 4°C for 1 h. Membrane pellets were resuspended in breakage buffer. The soluble fraction of the cells, and the supernatant fraction were concentrated using Amicon centrifugation, such that all three fractions were equal volume prior to analysis by immunoblotting. GroEL was assessed by immunoblot as a control for cytoplasmic proteins (rabbit anti-GroEL; Sigma, G6532).

Putnam E.E., Abellon-Ruiz J., Killinger B.J., Rosnow J.J., Wexler A.G., Folta-Stogniew E., Wright A.T., van den Berg B, & Goodman A.L. (2022). Gut Commensal Bacteroidetes Encode a Novel Class of Vitamin B12-Binding Proteins. mBio, 13(2), e02845-21.

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Western Blot Protein Analysis Protocol

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Bacterial samples were resuspended in 1× protein loading buffer and boiled at 98 °C for 8 min. 0.05 OD of proteins samples were loaded each lane. Proteins were transferred onto PVDF membranes (#10600023, Cytiva) for 45 min at 150 V in transfer buffer. Membranes were blocked for 1 h at room temperature in 1× TBST buffer with 5% (w/v) skim milk (#A600669-0250, Sangon), washed with TBST twice and incubated with monoclonal α-FLAG (Sigma-Aldrich #F1804-5MG; 1:10,000), α-SipC (1: 3,000) or α-GroEL (Sigma-Aldrich #G6532; 1:10,000) antibodies for 1 h at room temperature. After three TBST washes, membranes were incubated with secondary α-mouse or α-rabbit HRP-linked antibodies (Sangon #D110087 or #D110058; 1:10,000) for 1 h at room temperature. Chemiluminescence was developed using the high sensitive ECL luminescence reagent (#C500044-0100, Sangon), and then visualized on ChemiScope 6000SE and quantified using ImageJ Software.

Zhang J., Zhang S., Zhou W., Zhang X., Li G., Li R., Lin X., Chen Z., Liu F., Shen P., Zhou X., Gao Y., Chen Z., Chao Y, & Wang C. (2024). A widely conserved protein Rof inhibits transcription termination factor Rho and promotes Salmonella virulence program. Nature Communications, 15, 3187.

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EPEC Effector Secretion Quantification

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EPEC effector secretion was determined as described previously (Cameron et al., 2018 (link)). Briefly, apical media were centrifuged at 4000 g, 4°C for 10 min. Supernatants were filter sterilized, concentrated 8-fold using Amicon Ultra-4 centrifugal filter units (10 MWCO, Millipore), and 5× reducing sample buffer (RSB) was added. Bacterial pellets were suspended in 1× RSB. After denaturation at 95°C for 5 min, proteins were separated in 12% SDS-polyacrylamide gels (Mini-PROTEAN Tetra Cell, Bio-Rad) for 60 min at 150 V, 100 mA, 10 W. Proteins were transferred to PVDF membranes (Amersham™) by wet blotting at 100 V constant for 60 min. Membranes were blocked in 3% (w/v) bovine serum albumin in Tris-buffered saline with 0.05% (v/v) Tween-20 for 60 min and probed with rabbit anti-EspB (1:1000; Gad Frankel, Imperial College London, UK) (Knutton et al., 1998 (link)) or rabbit anti-E. coli GroEL (1:50,000; G6532, Sigma-Aldrich) overnight at 4°C. Blots were subsequently incubated with HRP-conjugated goat anti-rabbit IgG (1:200,000; Sigma-Aldrich) for 45 min. Membranes were developed using enhanced chemiluminescence (Pierce™) and imaged with a G:Box Chemi XRG Imager (Syngene). ImageJ Fiji software (https://imagej.net/software/fiji/) was used for densitometric analysis of imaged blots. EspB band intensities were normalized according to signals of the housekeeper protein GroEL.

McGrath C.J., Laveckis E., Bell A., Crost E., Juge N, & Schüller S. (2022). Development of a novel human intestinal model to elucidate the effect of anaerobic commensals on Escherichia coli infection. Disease Models & Mechanisms, 15(4), dmm049365.

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Measuring TpxBt Levels under H2O2 Stress

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To measure the levels of TpxBt in the presence of H₂O₂, overnight cultures of WT and TpxBt-HIS strains were diluted 250-fold into 15 mL of TYG and grown until mid exponential phase (OD₆₀₀ = 0.3-0.4). H₂O₂ was added at a final concentration of 100 μM, and 1.5 mL of culture was collected into microcentrifuge tubes and precipitated with the addition of 100% trichloroacetic acid (TCA) to a final of 13%. Cultures were collected before and 10min, and 30min after the addition of H₂O₂. Protein was precipitated on ice for 20 hours, washed with 1 mL of ice-cold acetone, and resuspended in 100 μL of 1X protein loading buffer. His-tagged proteins were detected using a 1:2000 dilution of HIS antibody (mouse) GenScript, #A00186-100 and a fluorescent secondary antibody (Goat anti-mouse IgG DyLight 680 Conjugated (Invitrogen, #35519)). GroEL protein levels were measured using a 1:5,000 dilution of anti-GroEL rabbit antibody (Sigma-Aldrich Cat# G6532, RRID:AB_259939) and a fluorescent secondary antibody (Donkey anti-rabbit IRDye 680LT (Li-Cor, #926-68023)). Fluorescence was detected using an Amersham Imager 600 instrument with an excitation wavelength 630 nm and captured by the Cy5 filter. Western blots were quantified using ImageJ software with background subtraction, each band was normalized to GroEL loading control.

Gebbia J.A., Coleman J.L, & Benach J.L. (2001). Borrelia Spirochetes Upregulate Release and Activation of Matrix Metalloproteinase Gelatinase B (MMP-9) and Collagenase 1 (MMP-1) in Human Cells. Infection and Immunity, 69(1), 456-462.

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