Neurons were lysed in ice-cold 1× RIPA buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% Na-deoxycholate, 0.1% SDS, and 1% NP-40) and supplemented with 1:100 protease inhibitor cocktail (Sigma-Aldrich). Lysed neurons were sheared by sonication, cell debris pelleted at 15000 rpm for 5 min at 4°C, and the clarified supernatant transferred to a prechilled 1.7 ml microcentrifuge tube. Total cell extracts were denatured at 95°C for 5 min, using either home-made 5× Laemmli buffer, BOLT 4× Sample buffer (Life Technologies) and BOLT 10× reducing agent (Life Technologies), 2×-, or 4× Laemmli sample buffer (both from Bio-Rad). To obtain synaptic fractions, cell pellets were treated with 1 ml ice-cold Syn-PER reagent (Thermo Scientific catalogue number 87793) and supplemented with 1:100 protease inhibitor cocktail (Sigma-Aldrich). Neurons were incubated on ice for 10 min, gently triturated 10 times, centrifuged at 1200 × g for 10 min at 4°C, and supernatant was transferred to ice-cold microcentrifuge tubes, then recentrifuged at 15,000 × g for 20 min at 4°C. The pellet was then resuspended in 20 µl 2× Laemmli sample buffer (Bio-Rad). Acid-extracted histone fractions were prepared using a protocol described previously (Shechter et al., 2007 (link)). Histone pellets were denatured using 2× Laemmli sample buffer (Bio-Rad).
Laemmli sample buffer
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Laemmli sample buffer is a commonly used buffer solution for protein sample preparation in SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) analysis. It helps denature and solubilize proteins, allowing for their separation and detection based on their molecular weight.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (111 mentions)
Ripa buffer, by Thermo Fisher Scientific (87 mentions)
Pvdf membrane, by Bio-Rad (76 mentions)
Trans blot turbo transfer system, by Bio-Rad (67 mentions)
Nitrocellulose membrane, by Bio-Rad (65 mentions)
2 mercaptoethanol, by Merck Group (62 mentions)
β mercaptoethanol, by Merck Group (60 mentions)
Chemidoc mp imaging system, by Bio-Rad (58 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (58 mentions)
Image lab software, by Bio-Rad (57 mentions)
Clarity western ecl substrate, by Bio-Rad (56 mentions)
Mini protean tgx precast gel, by Bio-Rad (56 mentions)
Pvdf membrane, by Merck Group (55 mentions)
Protease inhibitor cocktail, by Roche (54 mentions)
Protease inhibitor cocktail, by Merck Group (53 mentions)
Mini protean tgx gel, by Bio-Rad (41 mentions)
Chemidoc imaging system, by Bio-Rad (40 mentions)
2 mercaptoethanol, by Bio-Rad (37 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (34 mentions)
Ripa buffer, by Merck Group (33 mentions)
2 047 protocols using laemmli sample buffer
1
Neuronal Protein Extraction and Fractionation
2
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 0.5% Tween-20, 1% Nonidet P-40, and protease inhibitors for 20 min on ice. Lysates were cleared by centrifugation, and soluble protein was used for immunoprecipitation or mixed with 2 Laemmli Sample Buffer (Bio-Rad) and incubated for 7 min at 96 °C and analyzed by Western blot. For immunoprecipitation, protein extracts were incubated with GFP-Trap beads (ChromoTek) at 4 °C for 120 min. Beads were washed five times with lysis buffer and incubated with 2 Laemmli Sample Buffer (Bio-Rad) for 7 min at 96 °C. In case of phosphatase treatment, washed beads after IP were incubated with 10 U of FastAP (Fermentas) in 1 FastAP buffer at 37 °C for 30 min, pelleted, and incubated with Laemmli Sample Buffer. Proteins were resolved in 4–12% Bis-Tris or 3–8% Tris–acetate gels (Life Technologies), transferred to 0.45- m nitrocellulose membrane (Bio-Rad), and immunoblotted.
3
Protein Extraction from Lmo10403s Strains
4
Cell Lysis and Western Blot Preparation
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AsE01 and S2 cells were grown and transfected as described above. After 14 h of incubation, cells were harvested in sterile PBS and centrifuged (30 min, 2716 rcf, 10°C
for AsE01; 10 min, 244 rcf 10°C for S2). Cell pellets were washed with 1 ml ice-cold PBS and centrifuged again as above. The pelleted cells were lysed in ice-cold Pierce RIPA buffer (Thermo Fisher Scientific) supplemented with 1x SIGMAFAST Protease Inhibitor (Sigma-Aldrich). The cell mixture was vigorously pipetted, supplemented with 4x Laemmli sample buffer (Bio-Rad) containing 50 mM DTT and boiled for 5 min at 95°C. For transgenic flies, 15 larvae (heterozygous third instar) from each line were homogenized in 2x Laemmli sample buffer (Bio-Rad) containing 355 mM 2mercaptoethanol (Sigma-Aldrich). Homogenates were incubated at 95°C for 5 min then centrifuged at 10.000 g for 5 min. The supernatant was stored at -20°C.
for AsE01; 10 min, 244 rcf 10°C for S2). Cell pellets were washed with 1 ml ice-cold PBS and centrifuged again as above. The pelleted cells were lysed in ice-cold Pierce RIPA buffer (Thermo Fisher Scientific) supplemented with 1x SIGMAFAST Protease Inhibitor (Sigma-Aldrich). The cell mixture was vigorously pipetted, supplemented with 4x Laemmli sample buffer (Bio-Rad) containing 50 mM DTT and boiled for 5 min at 95°C. For transgenic flies, 15 larvae (heterozygous third instar) from each line were homogenized in 2x Laemmli sample buffer (Bio-Rad) containing 355 mM 2mercaptoethanol (Sigma-Aldrich). Homogenates were incubated at 95°C for 5 min then centrifuged at 10.000 g for 5 min. The supernatant was stored at -20°C.
5
Protein Extraction and Quantification for Mass Spectrometry
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30 µg of cell culture media were first measured by BCA assay (Thermo Fisher Scientific Inc., Waltham, USA) and then mixed with 7.5 µl of 4x Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, USA) in a final volume 30 µl. 30 µl of cell culture media proteins with final 1x Laemmli Sample Buffer were boiled at 70°C for 10 min and sonicated at 4°C for 5 min. The sonicated protein extracts were further sent to the proteomics core facility (Bavarian Center for Biomolecular Mass Spectrometry).
25 µl EVs were first mixed with 25 µl 2x RIPA buffer (Abcam plc, Cambridge, UK; ab156034) and stored at -80°C for overnight incubation. Next, samples were boiled at 70°C for 10 min and sonicated at 4°C for 5 min. The sonicated protein extracts were centrifuged at 10,000 rcf at 4°C for 30 min. Protein concentration was measured by performing a BCA assay (Thermo Fisher Scientific Inc., Waltham, USA). 2 µg of EV proteins were further mixed with ddH2O and 5 µl of 4x Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, USA) with 2-mercaptoethanol (Merck KGaA, Darmstadt, Germany) in a final volume of 20 µl. 20 µl of EV proteins with 1x Laemmli Sample Buffer were sent to the proteomics core facility (Bavarian Center for Biomolecular Mass Spectrometry).
25 µl EVs were first mixed with 25 µl 2x RIPA buffer (Abcam plc, Cambridge, UK; ab156034) and stored at -80°C for overnight incubation. Next, samples were boiled at 70°C for 10 min and sonicated at 4°C for 5 min. The sonicated protein extracts were centrifuged at 10,000 rcf at 4°C for 30 min. Protein concentration was measured by performing a BCA assay (Thermo Fisher Scientific Inc., Waltham, USA). 2 µg of EV proteins were further mixed with ddH2O and 5 µl of 4x Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, USA) with 2-mercaptoethanol (Merck KGaA, Darmstadt, Germany) in a final volume of 20 µl. 20 µl of EV proteins with 1x Laemmli Sample Buffer were sent to the proteomics core facility (Bavarian Center for Biomolecular Mass Spectrometry).
6
Outer Membrane Vesicle Characterization
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OMV solutions corresponding to 7.5 µg of each type of OMV lipids were mixed with 4 µL of 4X Laemmli sample buffer (Bio-Rad). Whole cell extracts of each bacterial clone (corresponding to 3 µg of proteins) were mixed with 4 µL of 4X Laemmli sample buffer (Bio-Rad). Following SDS-PAGE, the proteins and associated LPS were transferred from the polyacrylamide gel to a nitrocellulose membrane (Bio-Rad) using a Trans-Blot turbo apparatus (Bio-Rad) according to the manufacturer’s recommendations. The membrane was blocked for 30 min in 5% non-fat dry milk in TBS-T [50 mM Tris, 200 mM NaCl, pH 7.5, 0.1% Tween-20], then incubated overnight in 2.5% non-fat dry milk in TBS-T containing either mouse monoclonal anti E. coli LPS IgGs (clone 2D7/1 (ab35654), Abcam, Cambridge, United Kingdom) diluted 1/2000 or rabbit anti-OmpA polyclonal antibodies (Epigentek, Farmingdale, United States) diluted 1/2000. After two washes of 10 min each in TBS-T, the membranes were incubated for 3 h, respectively, in 2.5% non-fat dry milk in TBS-T containing anti-mouse polyclonal antibodies coupled to horse radish peroxidase [HRP] and diluted 1/10,000 or containing HRP-conjugated anti-rabbit IgG F(ab’)2 fragment (Sigma) diluted 1/10,000. After washes in TBS-T, the membranes were revealed by ECL using a Clarity Western substrate kit (Bio-Rad) and photographed using a ChemiDoc apparatus (Bio-Rad).
7
Immunoprecipitation of NLRP3 and Flag-tagged Proteins
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Cardiac tissue protein lysates were obtained using immunoprecipitation (IP) lysis buffer containing protease inhibitor (Absin Bioscience, Shanghai, China). Lysates were then incubated with Protein A/G agarose beads (Absin Bioscience, Shanghai, China). Samples were immunoprecipitated using anti‐NLRP3 antibody (15101,CST) or normal rabbit immunoglobulin G (2729S, CST) overnight at 4 °C. Immunoprecipitates were washed with lysis buffer 3 times. Laemmli sample buffer (Bio–Rad) was then added, and samples were boiled, separated by SDS–PAGE, and immunoblotted.
The granulocyte cell line NB4 was transfected with the NLRP3‐Flag plasmid. Then, cell lysates were obtained via IP lysis buffer containing protease inhibitor (Absin Bioscience, Shanghai, China). Cell lysates were then incubated with Protein A/G agarose beads (Absin Bioscience, Shanghai, China). Then, samples were immunoprecipitated with anti‐Flag antibody (F1804, Sigma) or normal rabbit immunoglobulin G (2729S, CST) overnight at 4 °C. Immunoprecipitates were washed with lysis buffer 3 times and eluted with Flag peptide. Laemmli sample buffer (Bio–Rad) was then added, and samples were boiled, separated by SDS–PAGE, and immunoblotted.
The granulocyte cell line NB4 was transfected with the NLRP3‐Flag plasmid. Then, cell lysates were obtained via IP lysis buffer containing protease inhibitor (Absin Bioscience, Shanghai, China). Cell lysates were then incubated with Protein A/G agarose beads (Absin Bioscience, Shanghai, China). Then, samples were immunoprecipitated with anti‐Flag antibody (F1804, Sigma) or normal rabbit immunoglobulin G (2729S, CST) overnight at 4 °C. Immunoprecipitates were washed with lysis buffer 3 times and eluted with Flag peptide. Laemmli sample buffer (Bio–Rad) was then added, and samples were boiled, separated by SDS–PAGE, and immunoblotted.
8
Induction and Purification of V. alginolyticus T3SS
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To induce the V. alginolyticus T3SS secretion, overnight cultures were diluted 1:100 into the inducing TSB medium as described (with chloramphenicol) and were cultured at 30°C with shaking. When OD600 reached ∼0.6, IPTG (0.5 mM) was added to induce protein expression and then continued to grow for 5 h. Bacterial cells were pelleted by centrifugation (3200 × g, 10 min), and were then re-suspended in Laemmli sample buffer (Bio-rad). The supernatant was collected and filtered through a low-protein-binding filter with a 0.2-µm pore size (Acrodisc syringe filter; Pall Life Sciences). Trichloroacetic acid (TCA) precipitation was carried out as described previously [36 (link),37 (link)] with a minor modification. Briefly, TCA was added to a final volume of 20% and incubated in ice-water for 2 h. After centrifugation at 22,000 × g for 30 min at 4°C, the pellets were resuspended in pre-chilled acetone and washed twice. The final protein pellet was dried in air and re-suspended in Laemmli sample buffer (Bio-Rad). All samples were boiled for 8 min and analyzed by western blots (see below). The primary antibodies were monoclonal antibody to His tag (1:2000 dilution; Abcam) or polyclonal antibody to DnaK (1:1000 dilution) [38 (link)].
9
Protein Extraction and Immunoblotting
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Cells were seeded into 12-well tissue culture plates (Corning). After 24 h, cells were stimulated with LPS (O55:B5, Sigma-Aldrich) for 6 h; during the last 2 h of LPS stimulation, nigericin (InvivoGen) was added. Total protein lysates were prepared as previously described [41 (link)]; 4 × Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) with 10% 2-mercaptoethanol (Sigma-Aldrich) was added, and samples were frozen at −20 °C for subsequent immunoblot analysis. To extract proteins from cell supernatants, protein-binding StrataClean resin (Agilent, Santa Clara, CA, USA) was added; the mixture was vortexed for 30 s and subsequently spun (5 min, 250× g, 4 °C). StrataClean resin pellets were resuspended in 4× Laemmli sample buffer (Bio-Rad Laboratories) with 10% 2-mercaptoethanol (Sigma-Aldrich), and stored at −20 °C until being subjected to immunoblot analysis.
10
Flagellin Extraction and Analysis
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∆fliC (GSO1073) or WT (GSO983) cells harboring pBR*, pBR*-UhpU, pZE, pZE-MotR, pZE-MotR*, pZE-FliX or pZE-FliX-S were grown with shaking at 180 rpm in 5 ml of LB at 37 °C to OD600 ~1.0. Cell pellets collected by centrifugation were suspended in 5 ml of PBS and then heated at 65 °C for 5 min, followed by centrifugation to obtain the cell pellets and supernatants, which contained the cytoplasmic flagellin molecules and depolymerized flagellin monomers, respectively. The cell pellets were resuspended in the Laemmli sample buffer (Bio-Rad), normalized to the cell density. Proteins in the supernatants were precipitated by 10% trichloroacetic acid, resuspended in Laemmli sample buffer (Bio-Rad) and heated at 95 °C for 10 min.
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