Laemelli buffer

Manufactured by Bio-Rad

Sourced in United States

Laemelli buffer is a common buffer used in protein electrophoresis. It is designed to denature proteins and maintain their charge-to-mass ratio, allowing for separation based on molecular weight.

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Lab products found in correlation

β mercaptoethanol, by Merck Group (3 mentions) Protein a g, by Thermo Fisher Scientific (2 mentions) Biotin azide, by Thermo Fisher Scientific (2 mentions) A10044, by Thermo Fisher Scientific (2 mentions) Thymidine, by Merck Group (2 mentions) Odyssey blocking buffer pbs, by LI COR (2 mentions) Complete ultra tablet, by Roche (2 mentions) Tris glycine sds buffer, by Bio-Rad (2 mentions) Piece bca protein assay, by Thermo Fisher Scientific (2 mentions) Odyssey, by LI COR (2 mentions) Gapdh, by Merck Group (2 mentions) Pierce ripa buffer, by Thermo Fisher Scientific (2 mentions) Tris glycine buffer, by Bio-Rad (2 mentions) Phostop, by Roche (2 mentions) Tris glycine gel, by Bio-Rad (2 mentions) Protease inhibitory cocktail, by Roche (1 mentions) Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions) Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions)

13 protocols using laemelli buffer

Immunoprecipitation of Mouse Cortex Proteins

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Mouse cortices (3–5 months of age) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, with protease inhibitory cocktail from Roche, Basel, Switzerland) and solubilized for 1 h at 4 °C. Solubilized material was centrifuged at 20 000 g for 10 min at 4 °C and the supernatant was precleared with protein A/G (Thermo Fisher Scientific) for 30 min. Proteins in the precleared supernatant were then immunoprecipitated with 3 μg of antibody overnight at 4 °C, followed by a 1 h incubation with protein A/G the following day. Beads were then washed 3 times with IP buffer before adding 2x Laemelli buffer (Biorad, Hercules, CA, USA). Samples were analyzed by SDS-PAGE and western blotting using standard methods.

Gao R., Zaccard C.R., Shapiro L.P., Dionisio L.E., Martin-de-Saavedra M.D., Piguel N.H., Pratt C.P., Horan K.E, & Penzes P. (2019). The CNTNAP2-CASK complex modulates GluA1 subcellular distribution in interneurons. Neuroscience letters, 701, 92-99.

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Protein Extraction and Immunoblotting Protocol

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To prepare protein lysates, harvested cells were resuspended in 150 μl RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5%, 0.1% sodium dodecyl sul-fate) supplemented with protease inhibitor cocktail (#P-8340, Millipore Sigma) and phosphatase inhibitor cocktails 2 and 3 (#P5726 and #P0044, Millipore Sigma). Following tip sonication on ice for 2 × 10 s bursts, protein concentration was determined by Bradford assay with Biorad reagent (#50000006) from Biorad (Hercules, CA) and samples were combined with 2x Laemelli buffer (#1610737, Biorad) containing 5% β-mercaptoethanol (#M6250, Millipore Sigma). Equal protein amounts (10–20 μg) were separated by 4%–20% SDS-PAGE and transferred to nitrocellulose membrane by wet transfer using standard immunoblotting protocols. Membranes were blocked (5% milk in 0.1% tween PBS) and probed with antibodies (various concentrations in 2% bovine serum albumin in 0.1% tween PBS) overnight at 4C. For developing, membranes were washed (3 × 10 min, 0.1% Tween PBS), incubated at room temperature for 1 h in HRP-Conjugated Secondary antibody (1:5000, 2% bovine serum albumin in 0.1% tween PBS; Jackson Laboratories), washed (3 × 10 min, 0.1% Tween PBS), and developed by enhanced chemiluminescence (#32109) from ThermoFisher Scientific (Waltham, MA). Information on the primary antibodies used is shown above.

Linzer R.W., Guida D.L., Aminov J., Snider J.M., Khalife G., Buyukbayraktar A.B., Alhaddad C., Resnick A.E., Wang P., Pan C.H., Allopenna J.J, & Clarke C.J. (2022). Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival downstream of HER2-driven glucose uptake and metabolism. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(10), e22558.

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Exosome Isolation and Protein Analysis

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Pooled human serum (Valley Biomedical, Winchester, VA, catalog #HS1004P) and six commercially available individual donor samples (BioreclamationIVT, NY, Item #HMSRM) were used for exosome isolation. We used three exosome isolation kits for this study: miRCURY^TM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuick^TM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA). To isolate exRNA, miRCURY RNA Isolation Kit–Cell & Plant (Exiqon, Woburn, MA) was used. RIPA lysis and extraction buffer (GBiosciences, St. Louis, MO), 2xLaemelli buffer (Bio-Rad, Hercules, California), and 100x halt protease inhibitor EDTA-free cocktail (Thermo Scientific, Grand Island, NY) were used to prepare protein samples for western blot analysis. Rabbit monoclonal anti-CD63 antibody was from Abcam (Cambridge, MA), and rabbit polyclonal anti-CD9 antibody was purchased from Santa Cruz (Dallas, Texas). IRDye 800CW-conjugated goat anti-rabbit secondary antibody was from LI-COR (Lincoln, Nebraska).

Helwa I., Cai J., Drewry M.D., Zimmerman A., Dinkins M.B., Khaled M.L., Seremwe M., Dismuke W.M., Bieberich E., Stamer W.D., Hamrick M.W, & Liu Y. (2017). A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents. PLoS ONE, 12(1), e0170628.

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Cellular Protein Extraction and Analysis

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For cellular protein extraction, cells were scraped in 0.75% SDS and lysed by sonication. Bradford reagent (Bio-Rad, Hercules, CA, USA) was used to determine protein concentration before immunoblotting. Lysates were mixed with equal volumes of 2x Laemelli buffer (Bio-Rad) and boiled for 5 min. The protein was separated by SDS-PAGE using the Criterion gels (Bio-Rad) and immunoblotted as described previously.^{51 (link)}

Shamseddine A.A., Clarke C.J., Carroll B., Airola M.V., Mohammed S., Rella A., Obeid L.M, & Hannun Y.A. (2015). P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest. Cell Death & Disease, 6(10), e1947-.

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Western Blot Analysis of Bacterial Proteins

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Bacteria for Western blots were grown overnight in TSB with or without dextrose. The following morning 1 mL of culture was centrifuged at 13,000 × g for 1 min, the supernatant was removed, and was heat inactivated at 70°C for 10 min. The supernatants were diluted 1:4 with Laemelli buffer (Bio-Rad) with 2-mercaptoethanol (Fischer Scientific) and boiled at 99 C for 10 min. 40 μL of boiled supernatant was loaded onto a Mini Protean TGX 12% gel (Bio-Rad) and run on a Mini Trans-Blot apparatus (Bio-Rad) for 90 min at 150 V with a Chameleon Duo Pre-Stained Protein ladder (LI-COR). The gel was transferred to a supported nitrocellulose 0.22 μm membrane (Bio-Rad) for 60 min at 100 V. The membranes were blocked for 4 h with Intercept protein-free blocking buffer (LI-COR), and then incubated overnight with the Abcam mouse MAb anti-alphahemolysim antibody (ab190467) at 1 μg/mL. The following day, the membranes were washed for 10 min, then 5 min, then 5 min with PBS 0.05% Tween (Sigma-Aldrich). The membranes were then stained with the secondary antibody IR Dye 800CW Donkey anti-mouse (LI-COR) diluted in donkey serum (Sigma-Aldrich) for 1 h, then washed as above. The membranes were imaged on a LI-COR Odyssey machine with 7 (800) and 2 (700) gains on the fluorescent channels.

Stephens A.C., Thurlow L.R, & Richardson A.R. (2022). Mechanisms Behind the Indirect Impact of Metabolic Regulators on Virulence Factor Production in Staphylococcus aureus. Microbiology Spectrum, 10(4), e02063-22.

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Profiling Proteins at DNA Replication Forks

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Analysis of proteins associated with DNA replication forks was performed using the iPOND method. HeLa cells were pulse labelled with 10 μM EdU (A10044, Molecular probes, USA) for 15 minutes. For the pulse-chase experiment, the EdU was removed, washed and replaced with media containing 10 μM thymidine (T9250, Sigma, St Louis, MO) for 30 and 60 minutes. The cells were then fixed with 1% formaldehyde for 20 minutes and quenched with 0.125 mM glycine (pH 7) for 5 minutes. Cell pellets were suspended in 0.25% Triton X-100 in PBS, washed and incubated in click reaction buffer (2 mM CuSO4, 10 mM biotin azide (B10184, Invitrogen, Eugene, OR) and 10 mM sodium ascorbate) for 2 hours. A no click reaction sample that did not include biotin azide was used as a negative control. Nascent DNA was conjugated to biotin using click chemistry, and Streptavidin beads (69203, Novagen, USA) were used to capture the biotin conjugated DNA-protein complexes. The eluted proteins were either sent to MS Bioworks LLC, Ann Arbor, MI for proteome analysis or suspended in Laemelli buffer (161-0737, Biorad, USA) and analyzed by western blot.

Mani C., Tripathi K., Luan S., Clark D.W., Andrews J.F., Vindigni A., Thomas G, & Palle K. (2020). The Multifunctional Protein PACS-1 is Required for HDAC2 and HDAC3 Dependent Chromatin Maturation and Genomic Stability. Oncogene, 39(12), 2583-2596.

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Quantitative Analysis of Synaptic Proteins

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Snap frozen prefrontal cortex tissues NSRL 16B cohorts were hand homogenized in Pierce RIPA buffer (Thermo Scientific, #89900) with cOmplete ULTRA tablets (Roche, #05892791001) and PhoSTOP (Roche, #04906837001) protease inhibitors for whole cell lysis. Debris was removed via centrifugation and the protein concentration of the remaining homogenate was measured using the Piece BCA Protein Assay (Thermo Scientific, #23225) following the manufacturer’s instructions. 20 ug of protein in Laemelli buffer (BioRad, #1610747) with beta mercaptoethanol (Sigma, M-3148) was boiled at 100 °C, loaded into a precast 4–15% Tris Glycine gel (BioRad, #5671084), and run using Tris/Glycine/SDS buffer (BioRad, 1610772) at 150 V. Protein was transferred onto a nitrocellulose membrane (BioRad, 1620168) using Tris/Glycine buffer (BioRad, 1610771) at 100 V. Membranes were blocked using Odyssey Blocking Buffer (PBS) (LI-COR, 92740000) and incubated for a minimum of 14 hours with either synapsin 1 (1:2000 in PBS, Millipore, #AB1543) or PSD95 (1:1000 in PBS, Abcam, #ab13552). Blots were washed using TBS and incubated for 60 minutes in their host-specific secondary antibody (LI-COR, #92632210-Mouse, #92632211-Rabbit). Blots were imaged using Odyssey (LI-COR) imaging system and quantified using the Image Studio software. All blots were normalized to GAPDH (Sigma, #G8795).

Krukowski K., Feng X., Paladini M.S., Chou A., Sacramento K., Grue K., Riparip L.K., Jones T., Campbell-Beachler M., Nelson G, & Rosi S. (2018). Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits. Scientific Reports, 8, 7857.

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Western Blot Analysis of Synaptic Proteins

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Snap frozen hippocampal tissues were hand homogenized in Pierce RIPA buffer (Thermo Scientific, #89900) with cOmplete ULTRA tablets (Roche, #05892791001) and PhoSTOP (Roche, #04906837001) protease inhibitors for whole cell lysis. Debris was removed via centrifugation and the protein concentration of the remaining homogenate was measured using the Piece BCA Protein Assay (Thermo Scientific, #23225) following the manufacturer’s instructions. 20 ug of protein in Laemelli buffer (BioRad, #1610747) with beta mercaptoethanol (Sigma, M–3148) was boiled at 100 °C, loaded into a precast 4–15% Tris Glycine gel (BioRad, #5671084), and run using Tris/Glycine/SDS buffer (BioRad, 1610772) at 150 V. Protein was transferred onto a nitrocellulose membrane (BioRad, 1620168) using Tris/Glycine buffer (BioRad, 1610771) at 100 V. Membranes were blocked using Odyssey Blocking Buffer (PBS) (LI-COR, 92740000) and incubated for a minimum of 14 h with either synapsin 1 (1:2000 in PBS, Millipore, #AB1543) or PSD95 (1:1000 in PBS, Abcam, #ab13552). Blots were washed using TBS and incubated for 60 min in their host-specific secondary antibody (LI-COR, #92632210-Mouse, #92632211-Rabbit). No bands were visible when secondary antibodies alone were added. Blots were imaged using Odyssey (LI-COR) imaging system and quantified using the Image Studio software. All blots were normalized to GAPDH (Sigma, #G8795).

Krukowski K., Grue K., Frias E.S., Pietrykowski J., Jones T., Nelson G, & Rosi S. (2018). Female mice are protected from space radiation-induced maladaptive responses. Brain, behavior, and immunity, 74, 106-120.

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Western Blot Analysis of p70S6 Kinase

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Cells were washed with PBS and analyzed for levels of whole protein or phosphorylated p70S6k1 by Western blot. Washed cells were lysed in RIPA buffer containing protease and a phosphatase inhibitor (Halt Protease Inhibitor Cocktail; Thermo Fisher Scientific, Inc., Waltham, MA). After debris was removed by centrifugation, Laemelli buffer (Bio‐Rad, Hercules, CA) was added 1:1 to the cell lysates. Lysates were boiled and then run on a 4%–15% precast gradient gel (Bio‐Rad) at 150 V at room temperature. The proteins were then electrophoretically transferred to a nitrocellulose membrane at 100 V for 1 hour at 4°C. Following transfer, the membrane was washed twice in Tris‐buffered saline with 0.1% Tween 20 (TBST), blocked for 1 hour at room temperature in 2% Bovine Serum Albumin, 0.5% sodium azide in TBST, and then incubated overnight at 4°C with antibody against p70S6k1 (ab9366; AbCam, Cambridge, MA) or phosphorylated p70S6k1 at T389 (ab2571; AbCam). Anti‐β‐actin (Sigma‐Aldrich) was used as a loading control. After incubation with the primary antibody, the membranes were incubated with anti‐rabbit horseradish peroxidase antibody (GE Healthcare Bio‐Sciences, Pittsburgh, PA) for 1 hour at room temperature and then washed extensively with TBST. Detection was performed using Western Lightening ECL (Perkin‐Elmer, Waltham, MA).

Takeda K., Ning F., Domenico J., Okamoto M., Ashino S., Kim S., Jeong Y.Y., Shiraishi Y., Terada N., Sutherland E.R, & Gelfand E.W. (2018). Activation of p70S6 Kinase‐1 in Mesenchymal Stem Cells Is Essential to Lung Tissue Repair. Stem Cells Translational Medicine, 7(7), 551-558.

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Western Blot Protein Analysis Protocol

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At the completion of treatment times, cells were washed twice with phosphate-buffered saline and lysed in Laemelli buffer (Bio-Rad, Hercules, Calif). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk dissolved in 1× Tris-buffered saline, 0.1% Tween-20 (Sigma-Aldrich). Primary antibodies were dissolved in 4% bovine serum albumin (Sigma-Aldrich) in 1× Tris-buffered saline, 0.1% Tween-20. Secondary antibodies were prepared in 5% nonfat milk in 1× Tris-buffered saline, 0.1% Tween-20. Membranes were developed using Pierce ECL Chemiluminescent (Thermo Fisher Scientific, Rockford, Ill). Protein quantification was performed by densitometric analysis using ImageJ Software (National Institutes of Health, Bethesda, Md). Histograms depict mean expression of protein of interest relative to control protein with standard error of the mean.

Bennett D.T., Reece T.B., Foley L.S., Sjoberg A., Meng X., Fullerton D.A, & Weyant M.J. (2014). C-terminal tensin-like protein mediates invasion of human lung cancer cells and is regulated by signal transducer and activator of transcription 3. The Journal of thoracic and cardiovascular surgery, 149(1), 369-375.

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