Macrophage pellets were re-suspended in 30 μl of RIPA buffer (Beyotime, China). After incubation on ice for 1 h, the lysates were centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was mixed with sample buffer (Genscript, China) and incubated in boiling water for 10 min. The total cellular protein (30 μg/well) was separated by 10% SDS-PAGE. The separated proteins were transferred onto polyvinylidene difluoride membranes (PVDF, Millipore), which were then blocked overnight with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). The membranes were respectively incubated with primary antibodies to detect Arg-1 (1: 1000 Cell Signaling Technology) and MRC-1 (1: 1000 Proteintech). The housekeeping gene encoding beta-actin (1: 5000 Thermo Fisher Scientific) was used as an internal control. Finally, the target protein bands were visualized by ECL substrate (Advansta, China), and images were collected by the ChemiDoc XRS+ system (Bio-Rad, Inc.).
Sample buffer
Manufactured by GenScript
Sourced in United States
Sample buffer is a laboratory reagent used to prepare and stabilize biological samples for analysis. It typically contains a combination of salts, buffers, and other additives to maintain the integrity and properties of the sample. The core function of sample buffer is to create a suitable environment for the sample, facilitating downstream analytical processes.
Automatically generated - may contain errors
Lab products found in correlation
Broad pre stained protein marker, by GenScript (2 mentions)
Lds sample buffer, by GenScript (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl substrate, by Advansta (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Precast gel, by GenScript (1 mentions)
Pepsin, by Merck Group (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Mini protean tetra system, by Bio-Rad (1 mentions)
Sds page gel, by GenScript (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Coomassie brilliant blue, by Thermo Fisher Scientific (1 mentions)
Silverxpress silver staining kit, by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Transfer apparatus, by Bio-Rad (1 mentions)
6 protocols using sample buffer
1
Western Blot Analysis of M1/M2 Macrophage Markers
2
Protein Assay and Electrophoresis Protocol
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BCA protein assay kit was purchased from Thermo Scientific (Rockford, IL, USA). Pepsin and pancreatin were purchased from Sigma Aldrich (St. Louis, MO, USA). Broad pre-stained protein marker, sample buffer, LDS sample buffer and 4–20% precast gels were purchased from GenScript (Piscataway, NJ, USA).
3
SDS-PAGE Protein Separation and Visualization
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MPs (2 mg/ml) were dissolved in the sample buffer (GenScript, Piscataway, NJ, USA) or LDS sample buffer (GenScript, Piscataway, NJ, USA) at a ratio of 4:1 (v/v) and incubated at 95°C for 10 min. Then, mixture (15 μl) and broad pre-stained protein marker (8 μl, GenScript, Piscataway, NJ, USA) were loaded in each lane of the SDS-PAGE gels (GenScript, 4–20%, 15 wells). Electrophoresis was conducted by a Mini-Protean Tetra System (Bio-Rad Laboratories, Hercules, CA). The parameters of electrophoresis were set as 80 V for 30 min, followed by 100 V for 80 min at 4°C. After separation, gels were stained and images were acquired by an image scanner (GE Healthcare, Little Chalfont, SE).
4
Western Blot Protein Detection Protocol
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Samples containing 1.2X Sample Buffer (Genscript) were separated via SDS-PAGE using 4-12% Bis-Tris (Genscript) in 1X MES Running Buffer (Genscript) according to manufacturer’s instructions. Protein was transferred to nitrocellulose membranes (Bio-Rad) using the high molecular weight program of the Trans-Blot Turbo Transfer system (Bio-Rad) according to manufacturer’s instructions. Blocking was performed for 1 hour at room temperature with 100% LI-COR Odyssey Blocking Buffer. Primary antibody incubation (4°C, 16 hours) and secondary antibody incubations (22°C, 1 hour) were performed in 50% Odyssey Blocking Buffer/50% TBS + 0.1% Tween-20. Washes were conducted following primary and secondary incubations, 3 times 10 minutes, with excess TBS + 0.1% Tween-20. Blots were visualized and captured using the LI-COR Odyssey CL-X imaging system with LI-COR ImageStudio software. Densitometry analyses were performed using ImageStudio software.
5
Fractionation of Phosphorylated and Unphosphorylated α-Synuclein
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1, 0.5, 0.25, or 0.125
μg of pS129-α-syn and 1 μg of unphosphorylated
α-syn were fractionated using Sure PAGE 4–20% gradient
Bis-Tris gels (GenScript). Samples were prepared by mixing the protein
solution with sample buffer (GenScript) and heated at 95 °C for
10 min. Gels were stained in 0.1% (w/v) Coomassie Brilliant Blue (Thermo
Fisher Scientific) in 40% (v/v) methanol and 10% (v/v) acetic acid
for 1 h at RT and then destained in the same solution excluding Coomassie
Brilliant Blue. Silver-staining was performed using the SilverXpress
silver staining kit (Invitrogen) following the manufacturer’s
protocol.
μg of pS129-α-syn and 1 μg of unphosphorylated
α-syn were fractionated using Sure PAGE 4–20% gradient
Bis-Tris gels (GenScript). Samples were prepared by mixing the protein
solution with sample buffer (GenScript) and heated at 95 °C for
10 min. Gels were stained in 0.1% (w/v) Coomassie Brilliant Blue (Thermo
Fisher Scientific) in 40% (v/v) methanol and 10% (v/v) acetic acid
for 1 h at RT and then destained in the same solution excluding Coomassie
Brilliant Blue. Silver-staining was performed using the SilverXpress
silver staining kit (Invitrogen) following the manufacturer’s
protocol.
6
Cholerae Toxin Subunit B Expression
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V. cholerae (VC1) cells were grown with or without the presence of 100 μM tryptophol acetate at 30 oC for 16 h. Culture supernatants were obtained by centrifugation of these cultures at 1100xg for 10 min. The cell pellets lysed using × 5 Sample buffer (125 mM Tris, 0.25% BPB, 10% 2-Mercaptoethanol, 10% SDS, 50% Glycerol; GenScript, Piscataway, USA) for 5 min at 95 °C.
Protein concentrations for the analyzed cells extract samples were determined using Bio-Rad protein assay and then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer protocols (Bio-Rad). After incubation with 5% skim milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary goat anti-cholerae toxin sub unit B (1:500; 227040, Sigma-Aldrich) for 16 h at 4 °C. Membranes were washed three times for 10 min and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-goat secondary antibodies for 1 h at room temperature (A50-101P, Bethyl Laboratories, Montgomery, USA). Beta-actin used as loading control (1:1000, MP Biomedicals, Santa Ana, CA, USA). Blots were washed three times with TBST and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocols.
Protein concentrations for the analyzed cells extract samples were determined using Bio-Rad protein assay and then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer protocols (Bio-Rad). After incubation with 5% skim milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary goat anti-cholerae toxin sub unit B (1:500; 227040, Sigma-Aldrich) for 16 h at 4 °C. Membranes were washed three times for 10 min and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-goat secondary antibodies for 1 h at room temperature (A50-101P, Bethyl Laboratories, Montgomery, USA). Beta-actin used as loading control (1:1000, MP Biomedicals, Santa Ana, CA, USA). Blots were washed three times with TBST and developed with the ECL system (Amersham Biosciences) according to the manufacturer’s protocols.
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