Equivalent cell densities (normalized by A600) were pelleted by centrifugation (10,000 × g for 5 min). For Cu-treated cells, absorbance measurements were made at A500 to minimize interference by Cu. Cells were solubilized in Bugbuster (EMD Millipore) with added Benzonase and incubated at room temperature for 15 min. Samples were diluted 1:2 with 2× Tricine-SDS sample buffer (Novex) with 4% β-mercaptoethanol (BME). Lysates were incubated at 100°C for 5 min and then resolved on 16% Tricine gels (Novex) with Tricine-SDS sample buffer (Novex) at 125 V for 2.5 h. Resolved proteins were transferred to 0.2-μm nitrocellulose membranes which were then probed with polyclonal rabbit anti-Lpp (from the Silhavy laboratory collection of antiserum raised against denatured proteins, used at 1:800,000).
Tricine sds sample buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tricine SDS Sample Buffer is a laboratory reagent used to prepare samples for electrophoresis analysis. It contains Tricine, a zwitterionic buffer, and sodium dodecyl sulfate (SDS) to denature and solubilize proteins. The buffer aids in the separation and analysis of proteins in polyacrylamide gel electrophoresis (PAGE) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Tricine sds running buffer, by Thermo Fisher Scientific (2 mentions)
Bugbuster, by Merck Group (2 mentions)
Tricine gel, by Thermo Fisher Scientific (2 mentions)
Image studio lite software, by LI COR (1 mentions)
Novextm 10 20 tricine gel, by Thermo Fisher Scientific (1 mentions)
Sep pak c18 cartridge, by Waters Corporation (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Cellytic m lysis reagent, by Merck Group (1 mentions)
Tricine protein gels, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
F1804 200ug, by Merck Group (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
7 protocols using tricine sds sample buffer
1
Quantifying Outer Membrane Lipoprotein
2
Western Blot Analysis of Amyloid-β Oligomers
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Aβ42 aggregate samples were diluted with Tricine SDS Sample Buffer (Cat # LC1676, Thermo Fisher Scientific, Waltham, MA, USA) and separated on a 10–20% Tris-Tricine gel using Tricine SDS Running Buffer (Cat # LC1675, Thermo Fisher Scientific, Waltham, MA, USA). The separated bands were transferred onto a nitrocellulose membrane and detected with the mouse monoclonal anti-Aβ oligomer specific antibody NU-2 (1:4000; Klein’s lab) and the mouse monoclonal anti-β-Amyloid antibody 6E10 (1:1000; Cat # 803001, BioLegend, San Diego, CA, USA). The membrane was probed with s680RD Goat anti-Rabbit IgG Secondary Antibody (1:10,000; Cat # 926-68071, LI-COR Biosciences, Lincoln, NE, USA) and 800CW Goat anti-Mouse IgG Secondary Antibody (1:10,000; Cat # 926-32210, LI-COR Biosciences, Lincoln, NE, USA). The protein bands were quantified by the Image Studio Lite software (version 5.2, LI-COR Biosciences, Lincoln, NE, USA). Molecular weights were estimated using a prestained protein ladder from Bio-Rad (Hercules, CA, U.S.A.).
3
Peptide Characterization by Thioflavin T
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Chemically synthesized peptides (purity is >95%); Milli-Q water (Milli-Q system, Millipore, Bedford, MA); Dithiothreitol (DTT); Thioflavin T (ThT) (Sangon Bioengineering (Shanghai) Co., Ltd.; Tricine SDS Sample Buffer; SeeBlueTM Plus2 Prestained Standard; NovexTM 10–20% Tricine Gel; Tricine SDS Running Buffer (10×) (ThermoFisher SCIENTIFIC, United States).
4
Purification and Western Blot of AgRP
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Medium were collected after 48 hours culture of the cells. After centrifugation, the medium were applied to Sep-Pak C18 cartridges (Waters Corp., Milford, MA) pre-equilibrated with 0.9% saline. Cartridges were washed in saline and 5% CH3CN/0.1% trifluoroacetic acid (TFA) and eluted with 60% CH3CN/0.1% TFA. Eluates were lyophilized and reconstitute with Tricine SDS sample buffer with reducing agent (Life Technologies). Tricine SDS-PAGE and Western blot analysis were performed as described previously [19 (link)] using anti-AgRP antibody (1:2000).
5
GlpG Knockout Strain for TatA Substrate Analysis
6
Protease Stability of Murine Serum Peptides
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The peptides (HT-47, WRK-30, and dWRK-30) were mixed with pooled murine serum, obtained from C57BL6/N wildtype mice via cardiac puncture immediately after death by cervical dislocation (the procedure was approved by the local government authority LANUV under permit no. UniKöln_Anzeige§4.22.001), to a final concentration of 30 µM. In parallel, a control with water and pooled murine serum was prepared. Both mixtures were incubated at room temperature, and samples were taken at the time points indicated in Supplementary Table S3 . The samples were immediately added to a Novex Tricine SDS Sample Buffer containing a NuPage Sample Reducing Agent and incubated at 85 °C for 2 min. Boiled samples were stored at 4 °C. After the collection of all time points, samples were separated into 10–20% Tricine Gels. Gels with HT-47 and WRK-30 were subsequently transferred onto PVDF membranes (0.2 µm pore size) and immunoblot analysis with 1:200 dilutions of a custom-ordered and affinity-purified rabbit anti-WRK-30 (Eurogentec, Seraing, Belgium) antibody was performed. Gels with dWRK-30 were stained with Coomassie brilliant blue.
7
Western Blot Analysis of Transfected HEK293 Cells
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Forty-eight hours post transfection, the HEK293 cells were lysed using CelLytic M lysis reagent (Sigma-Aldrich). Clarified cell lysates (30 μl) were mixed with 2× Tricine SDS Sample Buffer (Novex, Thermo Fisher Scientific) and run on 10–20% Tricine Protein Gels (Novex, Thermo Fisher Scientific) in Tricine SDS Running Buffer (Novex, Thermo Fisher Scientific) at 125 V for 90 min. Proteins were transferred to polyvinylidene fluoride membrane (0.2 μm, Bio-Rad) at 100 mA for 2 h in Tris-Gly Transfer Buffer (Novex, Thermo Fisher Scientific) supplement with methanol (Sigma-Aldrich). Immunoblots were incubated with primary monoclonal anti-FLAG M2 antibody (1:1000, F1804-200UG, Sigma-Aldrich) and anti-β-actin (1:10,000, AM4302, Ambion, Thermo Fisher Scientific) overnight at 4 °C and then secondary anti-mouse IgG and horseradish peroxidase-linked antibody (1:5000, Cell Signaling) at room temperature for 2 h. Immunoblots were developed with Western ECL (Clarity, Bio-Rad). Full, uncropped versions of all blot images are provided in Supplementary Fig. 16 .
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