Laemmli sample loading buffer

Manufactured by Bio-Rad

Sourced in United States

Laemmli sample loading buffer is a solution used to prepare protein samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It contains the anionic detergent SDS, a reducing agent, and a tracking dye, which denature and solubilize proteins, allowing for their separation based on their molecular weight.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pvdf membrane, by Bio-Rad (3 mentions) Chemi lumi one super, by Nacalai Tesque (2 mentions) Ripa buffer, by Fujifilm (2 mentions) Anti sox2, by Cell Signaling Technology (1 mentions) Cell lysis buffer, by Cell Signaling Technology (1 mentions) Luminata crescendo western hrp substrate, by Merck Group (1 mentions) Anti tubulin, by Merck Group (1 mentions) Mini protean precast gel, by Bio-Rad (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Anti f4 80, by Hycult Biotech (1 mentions) Anti β actin, by Merck Group (1 mentions) Blocking one, by Nacalai Tesque (1 mentions) Las 1000 plus instrument, by Fujifilm (1 mentions) Immobilon p, by Merck Group (1 mentions) Precision plus protein dual color standard, by Bio-Rad (1 mentions) Phospho ampkα thr172, by Cell Signaling Technology (1 mentions) Immune blot polyvinylidene difluoride membrane, by Bio-Rad (1 mentions) Criterion tgx gradient gels, by Bio-Rad (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

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8 protocols using laemmli sample loading buffer

Western Blot Analysis of Protein Expression

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Tissue samples were homogenized in RIPA buffer (FUJIFILM) and the total protein concentration of each was measured using a BCA protein assay kit (Thermo Fisher Scientific). Protein lysate was mixed with Laemmli sample loading buffer (BioRad, CA, United States), and aliquots containing equal amounts of protein were separated by SDS-PAGE. The isolated proteins were transferred to PVDF membranes (BioRad), which were blocked using Blocking One (nacali tesque) and then incubated with primary antibodies. The target proteins were visualized using Chemi-Lumi One Super (nacali tesque). The antibodies used for immunoblotting are shown in Table 1. The target protein expression levels were quantified using Image J (NIH, MD, United States).

Miura I., Okada K., Ishii A., Warabi E., Watahiki T., To K., Shimano H., Ariizumi S, & Shoda J. (2022). p62/Sqstm1 rescue in muscle retards the progression of steatohepatitis in p62/Sqstm1-null mice fed a high-fat diet. Frontiers in Physiology, 13, 993995.

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SOX2 Protein Expression Analysis

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Cells were washed twice with PBS and lysed in a cold cell Lysis Buffer (Cell Signaling Technology, QLD, Australia) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) then sonicated for 10 s at 10 mA. For each sample, 15 µg of protein was mixed with Laemmli sample loading buffer (Bio-Rad, NSW, Australia) supplemented with the reducing agent dithiothreitol (DTT), denatured at 95 °C for 5 min, loaded into Mini-PROTEAN precast gels (Bio-Rad, NSW, Australia), and subjected to electrophoresis at 100 V. The gel was then transferred to PVDF membranes (Bio-Rad, NSW, Australia) and blocked with 5% skim milk. The membrane was incubated overnight at 4 °C with the primary antibodies, anti-SOX2 (Cell Signaling Technology, QLD, Australia, #23064),and anti-tubulin (Sigma-Aldrich, NSW, Australia, #T1568). The signal was detected with Luminata Crescendo Western HRP Substrate (Millipore, NSW, Australia) using the ChemiDoc MP Imaging System (Bio-Rad, NSW, Australia).

Gandhi N.S., Wang E., Sorolla A., Kan Y.J., Malik A., Batra J., Young K.A., Tie W.J., Blancafort P, & Mancera R.L. (2021). Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor. International Journal of Molecular Sciences, 22(17), 9354.

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Protein Extraction and Western Blotting

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Five thoraces were added to 75 µl of lysis buffer (62mM Tris pH7.5, 0.1% SDS with 1X Halt protease and phosphatase inhibitors). The samples were boiled for 5 min at 95°C and centrifuged to collect the supernatant. Laemmli sample loading buffer (cat no: 1610747; Bio-Rad) was added, and the samples were subjected to SDS-PAGE followed by western blot.

Vang S., Helton E.S., Guo Y., Burpee B., Rose E., Easter M., Bollenbecker S., Hirsch M.J., Matthews E.L., Jones L.I., Howze PH I.V., Rajasekaran V., Denson R., Cochran P., Attah I.K., Olson H., Clair G., Melkani G., Krick S, & Barnes J.W. (2024). O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis. Frontiers in Immunology, 15, 1387197.

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Quantification of Liver Protein Biomarkers

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Liver tissue specimens were homogenized in RIPA buffer (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and the total protein concentration of each sample was measured by a BCA protein assay kit (Thermo Fisher Scientific). Protein lysate was mixed with Laemmli sample loading buffer (BioRad, CA, USA), and equal amounts of protein were separated by SDS-PAGE. The isolated proteins were transferred to PVDF membranes (BioRad), which were blocked using Blocking One (Nacalai Tesque, Kyoto, Japan). Antibodies were obtained as follows: anti-NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) from Abcam (ab2346, Cambridge, UK), anti-lipopolysaccharide binding protein (LBP) from Proteintech (23559-1-AP, Tokyo, Japana), anti-F4/80 from Hycult Biotech (HM1066, Uden, Yhe Netherlamds,), anti-hexanoyl-lysine (HEL) from JaiCA, (MHL-021P, Shizuoka, Japnan), and anti-β-actin from Sigma (A5441, Deisenhofen, Germany). Target proteins were visualized using Chemi-Lumi One Super (Nacalai Tesque), and were quantified using ImageJ software (NIH, MD, USA).

Chihara K., Okada K., Uchida F., Miura I., Komine S., Warabi E., Takayama T., Suzuki H., Matsuzaka T., Ishibashi-Kanno N., Yamagata K., Yanagawa T., Bukawa H, & Shoda J. (2023). Macrophage specific restoration of the Nrf2 gene in whole-body knockout mice ameliorates steatohepatitis induced by lipopolysaccharide from Porphyromonas gingivalis through enhanced hepatic clearance. PLOS ONE, 18(10), e0291880.

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Western Blot Analysis of Enzyme Modifications

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After the enzyme (0.5 μM) was incubated with the quinones, 10 mM DTT and 4 × Laemmli sample loading buffer (Bio-Rad, CA, USA) were added to the reaction mixture. The resulting sample, along with Precision Plus Protein Dual Color Standard (Bio-Rad), was applied to a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) using 10% TGX™ FastCast™ acrylamide (Bio-Rad) and then migrated by electrophoresis. The proteins in the gels were transferred onto a PVDF membrane (Immobilon-P, Merck) via blotting. The membrane was blocked with EzBlock Chemi (ATTO Co., Tokyo, Japan) for 30 min at room temperature. Subsequently, it was incubated with an anti-3C-like protease (main protease) rabbit polyclonal antibody (1/4000), anti-TD-modified protein monoclonal antibody (1/2500), or an anti-Q5HIAA-modified protein monoclonal antibody (1/2500) in 0.05% Tween® 20 containing Tris-buffered saline, followed by the treatment of the corresponding secondary antibody-HRP conjugate (DAKO). The binding was visualized using Chemi-Lumi One Super (Nacalai Tesque, Inc.). The detection was carried out using a LAS1000 Plus instrument (Fujifilm, Tokyo, Japan). The experiment was repeated twice.

Kato Y., Sakanishi A., Matsuda K., Hattori M., Kaneko I., Nishikawa M, & Ikushiro S. (2023). Covalent adduction of serotonin-derived quinones to the SARS-CoV-2 main protease expressed in a cultured cell. Free Radical Biology & Medicine.

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Liver Protein Expression Analysis

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Mice liver tissue were lysed in Laemmli sample loading buffer (Bio-Rad, Hercules, CA, USA) supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Proteins were separated on 4%–20% Criterion TGX gradient gels (Bio-Rad) and transferred to Immune-Blot polyvinylidene difluoride membranes (Bio-Rad). The membranes were then probed with rabbit primary antibodies to phospho-AMPKα (Thr172; #2535; Cell Signaling Technology, Boston, MA, USA) and phospho-Akt (Ser475; #9271, Cell Signaling Technology) diluted 1:1000, and rabbit and mouse primary antibodies to, respectively, GAPDH (#5174, Cell Signaling Technology) and β-actin (#a5441, Sigma-Aldrich, St. Louis, MO, USA) diluted 1:5000. Horseradish peroxidase conjugated anti-rabbit IgG and anti-mouse IgG (Cell Signaling Technology) secondary antibodies were then added at 1:2000 dilution and proteins detected using Amersham ECL Select Western Blotting detection reagents (GE Healthcare UK Limited, Buckinghamshire, UK) with the aid of an FL1500 iBright imaging system (Invitrogen Life Technologies Corp., Carlsbad, CA, USA). All semiquantitative measurement of band intensity was performed using the ImageJ analysis software (US National Institutes of Health, Bethesda, MD, USA).

Yang C., Mwangi S.M., Balasubramaniam A., Li G., Merlin-Zhang O., Liu Y, & Srinivasan S. (2023). Treatment of Obesity Through Glial Cell-Derived Neurotrophic Factor Lipid Nanoparticle Delivery in Mice. Gastro hep advances, 3(1), 38-47.

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Surface Biotinylation of Membrane Proteins

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Surface biotinylation was performed using a protocol adapted from Huang et al [48 (link)]. Briefly, cells were incubated with external control, colchicine, or H₂O₂ treatment for 4 h. After treatment, cells were exposed to 1 mg/mL of EZ-link sulfo-NHS-LC-LC-biotin (ThermoScientific) and incubated at room temperature for 30 m. Cells were washed with PBS + 100 mM glycine three times, then lysed using CHAPS lysis & IP buffer (FIVEphoton Biochemicals, San Diego, CA) with protease (Roche, Risch-Rotkreuz, Switzerland), sonicated, and centrifuged at 4 °C for 10 min. Total protein concentrations were determined and normalized between samples. Supernatant was mixed with immobilized Neutravidin beads (ThermoScientific) and rotated at 4 °C overnight. Beads were washed with 4 °C PBS 3 times and then combined with 2× Laemmli sample loading buffer (Bio-Rad) supplemented with 5% 2-mercaptoethanol. Beads were incubated for 10 m at 65 °C to elute proteins. Eluted proteins were then run on a 4-15% TGX precast gradient gel (Bio-Rad) and then electrophoresed proteins were transferred to a PVDF membrane. The membrane was blocked with 10% skim milk in TBST, then stained with K_V4.2 & K_V4.3 anti-rabbit primary antibody (Santa Cruz, Cat. No. sc-28634) in TBST with 1% BSA followed by goat anti-rabbit secondary antibody (Santa Cruz, Cat. No. sc-2057) in 5% skim milk in TBST before exposure.

Drum B.M., Yuan C., Li L., Liu Q., Wordeman L, & Santana L.F. (2016). Oxidative stress decreases microtubule growth and stability in ventricular myocytes. Journal of molecular and cellular cardiology, 93, 32-43.

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Western Blot Protocol for Protein Detection

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RIPA buffer (SIGMA) was used to lyse transfected 293T cells. Cell lysates were mixed with 2× Laemmli sample loading buffer (BioRad), boiled, and loaded on native-PAGE gels. Subsequently, proteins were transferred to a PVDF membrane by electroblotting. Then the membrane was incubated for 1 h at room temperature in blocking buffer (5% nonfat dry milk in PBS). The blocked blot was exposed to the HIV-Ig or tubulin antibody in blocking buffer with constant mixing. After extensive washing, bound antibodies were detected by chemiluminescence using horseradish peroxidase-conjugated species-specific secondary antibodies as described by the manufacturer (GE Healthcare).

Afonin K.A., Viard M., Kagiampakis I., Case C.L., Dobrovolskaia M.A., Hofmann J., Vrzak A., Kireeva M., Kasprzak W.K., KewalRamani V.N, & Shapiro B.A. (2014). Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles. ACS Nano, 9(1), 251-259.

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