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Pierce lane marker reducing sample buffer

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The Pierce Lane Marker Reducing Sample Buffer is a solution used to prepare protein samples for gel electrophoresis. It contains reducing agents that help denature proteins, allowing for their separation based on molecular weight.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Complete edta free protease inhibitor cocktail, by Merck Group (2 mentions) Amersham ecl prime western blotting detection reagent, by GE Healthcare (2 mentions) Imagequant las 4000, by GE Healthcare (2 mentions) Mini protean tgx stain free protein gel, by Bio-Rad (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Pageblue protein staining solution, by Thermo Fisher Scientific (1 mentions) Pre stained protein ladder, by Thermo Fisher Scientific (1 mentions) Peroxidase labelled secondary antibody, by Santa Cruz Biotechnology (1 mentions) Immobilon p pvdf membrane, by Merck Group (1 mentions) Anti calnexin, by Cell Signaling Technology (1 mentions) Anti alix, by Abcam (1 mentions) Concentrator plus 5305 vacuum centrifuge, by Eppendorf (1 mentions) Anti tsg101, by BD (1 mentions) Anti cd81, by Santa Cruz Biotechnology (1 mentions) Anti cd63, by Santa Cruz Biotechnology (1 mentions) Clarity western ecl substrate, by Bio-Rad (1 mentions) Mini protean tgx stain free gel, by Bio-Rad (1 mentions) Gelcode blue safe protein stain, by Thermo Fisher Scientific (1 mentions)

22 protocols using pierce lane marker reducing sample buffer

1

SDS-PAGE Protein Separation and Visualization

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Protein samples (either 1 µg of recombinant proteins or 30 µg of cell lysates per lane) were separated by electrophoresis using 12% concentrating SDS polyacrylamide gels. The samples were prepared by adding Pierce Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific, 39000, Rockford, IL, USA) and boiling for 10 min. Prestained Protein Ladder (10 to 180 kDa, Thermo Fisher Scientific, 26616) was used as size standard. After the electrophoresis (Minigel-Twin 846-010-100 and Multigel 846-010-200, Biometra, Göttingen, Germany), gels were stained with PageBlue™ Protein Staining Solution (Thermo Fisher Scientific, 24620, Vilnius, Lithuania).
Stravinskiene D., Imbrasaite A., Petrikaite V., Matulis D., Matuliene J, & Zvirbliene A. (2019). New Monoclonal Antibodies for a Selective Detection of Membrane-Associated and Soluble Forms of Carbonic Anhydrase IX in Human Cell Lines and Biological Samples. Biomolecules, 9(8), 304.
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2

Characterization of Extracellular Vesicles

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EV sample was concentrated using Concentrator plus 5305 Vacuum Centrifuge (Eppendorf AG, Germany), and protein concentration was measured with Pierce BCA Protein Assay Kit (Thermo Scientific). Concentrated sEV preparations and lysate of HCT116 cells were lysed in Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific), heated for 5 min at 95 °C, and subjected to electrophoresis using 10% SDS-PAGE. Proteins were transferred to an Immobilon-P PVDF Membrane (Merck Millipore) and the excess protein binding sites on the membrane were saturated with 5% bovine serum albumin blocking buffer (1 × TBS, 0.1% Tween-20) for 1 h. The membrane was incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-CD81 (1:250, mouse, catalogue number sc166029), anti-CD63 (1:300, mouse, sc5275) from Santa Cruz Biotechnology, anti-Alix (1:20000, rabbit, ab186429) from Abcam, anti-TSG101 (1:200, mouse, 612696) from BD Biosciences, and anti-Calnexin (1:1000, rabbit, 2679) from Cell Signaling. After incubation, the membrane was washed three times with 5% TBS-Tween and then, incubated with peroxidase-labelled secondary antibody (Santa Cruz Biotechnology) for one hour. After three washes, immobilized proteins were detected utilizing Clarity Western ECL Substrate (Bio-Rad) and the UVITEC chemiluminescence imager (UVITEC Cambridge, UK).
Boudna M., Machackova T., Vychytilova-Faltejskova P., Trachtova K., Bartosova R., Catela Ivkovic T., Al Tukmachi D., Jugas R., Pifkova L., Orlickova J., Kotoucek J., Pavlikova M., Sachlova M., Bohovicova L., Stanek T., Halamkova J., Kiss I., Grolich T., Svoboda M., Kala Z., Souckova K, & Slaby O. (2024). Investigation of long non-coding RNAs in extracellular vesicles from low-volume blood serum specimens of colorectal cancer patients. Clinical and Experimental Medicine, 24(1), 67.
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3

SDS-PAGE Protein Sample Preparation

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For SDS–PAGE, protein samples were denatured by boiling them with Pierce Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific) for 10 min at 98 °C. After centrifuging the samples at 17,000 × g, the clear supernatant was loaded on a 4–20% Mini-PROTEAN TGX Stain-Free Gel (Bio-Rad Laboratories) including PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) as a molecular weight reference. The electrophoresis was run for 30 min at a constant voltage of 180 V before staining the gel with GelCode Blue Safe Protein Stain (Thermo Fisher Scientific).
Schmidt F.V., Schulz L., Zarzycki J., Prinz S., Oehlmann N.N., Erb T.J, & Rebelein J.G. (2023). Structural insights into the iron nitrogenase complex. Nature Structural & Molecular Biology, 31(1), 150-158.
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4

Optimization of Cell Culture Reagents

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Reagents were from the following manufacturers: 35‐mm glass‐bottomed dishes (MatTek, P35G‐1.0‐14‐C), CELLview™ dishes (Greiner Bio‐One 627 871), poly‐L‐lysine (Sigma‐Aldrich, P6282), collagen IV (Sigma‐Aldrich, C5533), penicillin, streptomycin and amphotericin B (Invitrogen, 15240‐096), gentamicin (Krka, d. d., Novo Mesto, Slovakia), RPMI‐1640 (Life Technologies, A1049101), DMEM (Life Technologies, 31966021), Neurobasal™ A (Life Technologies, 10888022 and 12349015 (without phenol red)), HBSS 1640 (Life Technologies, 14025050), BME (Life Technologies 41010‐109), Opti‐MEM I (Life Technologies, 31985‐047), B‐27® Supplement (50X, serum free, Life Technologies, 17504044), foetal bovine serum (Life Technologies, 10270106), horse serum (Life Technologies, 26050088), GlutaMAX™‐I (Life Technologies, 3505038) Lipofectamine® 2000 (Invitrogen, 11668‐019), Metafectene® (Biontex Laboratories GmbH, T040), DMSO (Sigma‐Aldrich, 472301), FCCP (Tocris, 0453), G418 (Sigma‐Aldrich, A1720), MG132 (Tocris, 1748), protease inhibitor cocktail cOmplete (Roche, 04693116001), protein G‐sepharose 4B beads (Invitrogen, 101243), DC Protein Assay reagent (Bio‐Rad, 500‐0111), Pierce Lane Marker Reducing sample buffer (Thermo Fisher Scientific, 39000).
Safiulina D., Kuum M., Choubey V., Gogichaishvili N., Liiv J., Hickey M.A., Cagalinec M., Mandel M., Zeb A., Liiv M, & Kaasik A. (2018). Miro proteins prime mitochondria for Parkin translocation and mitophagy. The EMBO Journal, 38(2), e99384.
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5

Analyzing Secreted Proteins by Western Blot

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An equal number of cells were seeded per sample in order to ensure equal sample loading for all supernatants analysed by Western blot. Thus, 750 000 cells per well were seeded when using 6-well plates, or they were seeded to 75–80% confluence in cell-culture flasks. Proteins from cell-free culture supernatants were concentrated using trichloroacetic acid (TCA) (Koontz, 2014 (link)). One hundred percent TCA was added to supernatants for a final concentration of 20%. Samples were vortexed and incubated on ice for 30 min. After incubation, samples were centrifuged at 800 × g and supernatants were discarded. The pellets were washed twice with 100% cold acetone and allowed to air-dry. Pellets were resuspended with Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific) and boiled at 95°C for 5 min. Samples were loaded and run on SDS-PAGE gels and transferred onto PVDF membranes. Membranes were blocked with 5% BSA for 1 h at RT, and then incubated with anti-TMS1 (Abcam), caspase-1 (Nathan), IL-1β (Abcam), anti-NLRP3 (Cell Signaling) or HMGB1 (Nathan) primary antibodies overnight at 4°C. After primary incubation, blots were washed and incubated with secondary goat anti-rabbit HRP (Millipore) for another hour. Finally, membranes were washed and exposed to Luminata Forte (Millipore) substrate. Images were acquired using ChemiDoc XRS+ system and analysed using NIH-ImageJ.
Bui F.Q., Johnson L., Roberts J., Hung S.C., Lee J., Atanasova K.R., Huang P.R., Yilmaz Ö, & Ojcius D.M. (2016). Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3 inflammasome-dependent secretion of IL-1β and the danger signals ASC and HMGB1. Cellular microbiology, 18(7), 970-981.
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6

Cell Lysis and Protein Quantification

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24 h after treatment, cells were washed briefly with PBS. Cells were lysed on ice in RIPA buffer (R0278-500ML, Sigma-Aldrich) supplemented with protease inhibitor (cOmplete™, EDTA-free protease inhibitor cocktail (1836170001, Sigma-Aldrich) and phosphatase inhibitor (Phos-STOP™ 4906837001, Sigma-Aldrich). Cells were scraped and the lysate was pipetted into a pre-chilled Eppendorf tube. Lysis was allowed to proceed for 20 min on ice and cells were centrifuged at 16000×g for 10 min at 4 ℃. Supernatant was transferred into a fresh tube and Pierce BCA Protein assay was performed (23225) according to the manufacturer’s instructions. Samples were brought to equal concentrations using addition of RIPA buffer, and Pierce™ Lane Marker Reducing Sample Buffer (39000, Thermo Fisher Scientific) was added to a final concentration of 1×. The samples were boiled for 5 min at 95 ℃.
Tziortzouda P., Steyaert J., Scheveneels W., Sicart A., Stoklund Dittlau K., Barbosa Correia A.M., Burg T., Pal A., Hermann A., Van Damme P., Moens T.G, & Van Den Bosch L. (2024). PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport. Acta Neuropathologica, 147(1), 41.
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7

Whole Cell Protein Extraction

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Cells were briefly washed with DPBS. Cells were scraped in DPBS and transferred to a pre-chilled Eppendorf tube. They were centrifuged for 5 min at 1000×g at 4 ℃. Pellets were then lysed in RIPA buffer (R0278-500ML, Sigma-Aldrich) supplemented with protease inhibitor (cOmplete™ EDTA-free protease inhibitor cocktail, 1836170001, Sigma-Aldrich) and phosphatase inhibitor (Phos-STOP™, 4906837001, Sigma-Aldrich). Lysis was allowed to occur on ice for 30 min. Samples were centrifuged at 14000×g for 10 min at 4 ℃ and the supernatant was collected. Pierce BCA Protein assay (23225) was performed. Samples were brought to equal concentrations, supplemented with Pierce™ Lane Marker Reducing Sample Buffer (39000, Thermo Fisher Scientific), to a final concentration of 1× and boiled for 5 min at 95 ℃.
Tziortzouda P., Steyaert J., Scheveneels W., Sicart A., Stoklund Dittlau K., Barbosa Correia A.M., Burg T., Pal A., Hermann A., Van Damme P., Moens T.G, & Van Den Bosch L. (2024). PP2A and GSK3 act as modifiers of FUS-ALS by modulating mitochondrial transport. Acta Neuropathologica, 147(1), 41.
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8

Methanol-Chloroform Protein Extraction

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Polysomal fractions (1 mL) or pellet/supernatant fractions from 30% sucrose cushioning (1/5th of total amount, adjusted to 260 µL volume) were processed for methanol/chloroform protein extraction. Briefly, 600 µL of methanol and 150 µL of chloroform were added to each sample and samples were vortexed. Then, 450 µL of deionized water were added to each sample and samples were vortexed again. Samples were centrifuged at 14,000 × g for 1 min at room temperature, and the resulting aqueous phase was removed without disrupting the underlying white ring (protein interface). Subsequently, 450 µL of methanol were added to each sample, samples were vortexed, and then centrifuged at 14,000 × g for 2 min at room temperature. After centrifugation, supernatants were removed and pellets air-dried. Finally, pellets were resuspended in deionized water supplemented with Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific #39000) to a final volume of 15 µL and either stored at −80°C or heated at 95°C and directly used for SDS-PAGE.
Minati L., Firrito C., Del Piano A., Peretti A., Sidoli S., Peroni D., Belli R., Gandolfi F., Romanel A., Bernabo P., Zasso J., Quattrone A., Guella G., Lauria F., Viero G, & Clamer M. (2021). One-shot analysis of translated mammalian lncRNAs with AHARIBO. eLife, 10, e59303.
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9

Quantitative Western Blot Assay for AHA

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Cell lysate or protein extracts obtained from sucrose gradient fractions were supplemented with Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific #39000), heated at 95°C for 10 min, and separated by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes, then membranes were blocked overnight at 4°C in 5% milk prepared in 1× Tris-Buffered Saline (TBS) − 0.1% Tween20 supplemented with dibenzocyclooctyne-PEG4-biotin conjugate (Sigma-Aldrich #760749; 50 µM final concentration). Membranes were washed three times in 1× TBS − 0.1% Tween20 for 10 min each, then incubated with Precision Protein StrepTactin-HRP Conjugate (BioRad #1610380; 1:1000 in 5% milk prepared in 1× TBS − 0.1% Tween20) for 1 hr at room temperature, then washed again. Membranes were subsequently developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare #RPN2236). Images were acquired through the ChemiDoc MP Imaging System. ImageJ software (v 1.45s) was used for quantitation of AHA signal intensities.
Minati L., Firrito C., Del Piano A., Peretti A., Sidoli S., Peroni D., Belli R., Gandolfi F., Romanel A., Bernabo P., Zasso J., Quattrone A., Guella G., Lauria F., Viero G, & Clamer M. (2021). One-shot analysis of translated mammalian lncRNAs with AHARIBO. eLife, 10, e59303.
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10

Western Blot Analysis of Ribosomal Proteins

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Aliquots of 10–20 µL of cell lysate or protein extracts obtained from sucrose gradient fractions were supplemented with Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific #39000), heated at 95°C for 10 min, and separated by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes, then membranes were blocked for 1 hr at room temperature in 5% milk prepared in 1× TBS − 0.1% Tween20. Membranes were subsequently incubated for 1 hr at room temperature with the following primary antibodies, diluted in 5% milk prepared in 1× TBS − 0.1% Tween20: anti-RPL26 (Abcam #ab59567; 1:2000), anti-RPS6 (Abcam #ab40820; 1:1000), and anti-beta-actin (Abcam #ab8227; 1:2000). Membranes were washed three times in 1× TBS − 0.1% Tween20 for 10 min each, then incubated with the appropriate HRP-conjugated secondary antibodies for 1 hr at room temperature and washed again as before. Membranes were then developed using either Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare #RPN2236) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific #34095), depending on signal intensities. Images were acquired through the ChemiDoc MP Imaging System. ImageJ software (v 1.45s) was used for quantitation of signal intensities of protein bands.
Minati L., Firrito C., Del Piano A., Peretti A., Sidoli S., Peroni D., Belli R., Gandolfi F., Romanel A., Bernabo P., Zasso J., Quattrone A., Guella G., Lauria F., Viero G, & Clamer M. (2021). One-shot analysis of translated mammalian lncRNAs with AHARIBO. eLife, 10, e59303.
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