Proteins samples were collected using radio immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s protocol. 4X LDS sample loading buffer (Life Technologies) and 5% beta-mercaptoethanol (Bio-Rad, Hercules, CA) were added to cell lysates, and these samples were denatured at 95°C for 8 min. After gel electrophoresis, membranes were blocked in 5% milk/TBS-T for 1h followed by incubation with primary antibody overnight. After washes and incubation with respective horseradish peroxidase-conjugated secondary antibodies (anti-mouse 1:10,000 or anti-rabbit 1:20,000) for 1 h, protein bands were visualized using the SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific, Waltham, MA) with Image-Quant LAS 4000 biomolecular imager (GE Healthcare Life Sciences, Pittsburgh, PA). The densities of protein bands were quantified using ImageJ.
Lds sample loading buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LDS sample loading buffer is a reagent used in laboratory analysis and research applications. It is designed to prepare samples for electrophoresis, a technique used to separate and analyze biomolecules such as proteins or nucleic acids. The buffer helps denature and solubilize the samples, ensuring consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (4 mentions)
Bradford protein assay kit, by Thermo Fisher Scientific (4 mentions)
Fluostar optima plate reader, by BMG Labtech (4 mentions)
Albumin standard, by Thermo Fisher Scientific (4 mentions)
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (4 mentions)
β mercaptoethanol, by Bio-Rad (3 mentions)
Sample reducing agent, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (2 mentions)
Lin28a tat, by Thermo Fisher Scientific (2 mentions)
Bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Nu page xcell surelock module, by Thermo Fisher Scientific (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Ube2d3, by R&D Systems (1 mentions)
Human e1, by R&D Systems (1 mentions)
Cnbr activated sepharose resin, by GE Healthcare (1 mentions)
29 protocols using lds sample loading buffer
1
Western Blot Protein Detection Procedure
2
Brain Tissue Homogenization and Fractionation
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Mice were euthanized by sodium pentobarbital and perfused with 20 mL PBS, 5mM EDTA (pH 7.4), collected aseptically and stored at −80°C for future analysis. 10% (wt/vol) brain homogenates were prepared as described previously [33 (link)]. Briefly, homogenization was performed on ice in PBS, pH 7.4, using glass/Teflon tissue grinders attached to a cordless 12V compact drill (Ryobi). The brains were ground at low speed until homogeneous and stored at -80°C in 1 ml aliquots.
For 2D, 200 μl of 10% brain homogenate was diluted 2-fold in PBS buffer supplied with proteinase inhibitors (cat# 1836145, Roche) and sonicated for 30 seconds in a water bath at 37°C. The sample was subsequently centrifuged at 16,000 g at 4°C for 30 minutes, the supernatant was discarded, and the pellet dissolved in 100 μl 1% Triton X-100 in PBS. 18 μl of brain material was mixed with 6 μl 4x LDS sample loading buffer (cat # NP0007, Life Technologies, Carlsbad, CA), incubated for 10 minutes at 95°C and subsequently used for SDS-PAGE or 2D gel electrophoresis.
For 2D, 200 μl of 10% brain homogenate was diluted 2-fold in PBS buffer supplied with proteinase inhibitors (cat# 1836145, Roche) and sonicated for 30 seconds in a water bath at 37°C. The sample was subsequently centrifuged at 16,000 g at 4°C for 30 minutes, the supernatant was discarded, and the pellet dissolved in 100 μl 1% Triton X-100 in PBS. 18 μl of brain material was mixed with 6 μl 4x LDS sample loading buffer (cat # NP0007, Life Technologies, Carlsbad, CA), incubated for 10 minutes at 95°C and subsequently used for SDS-PAGE or 2D gel electrophoresis.
3
LIN28A Binding Affinity Assay
4
GSDMD Ubiquitination by IpaH7.8
5
Nanobody-Human Serum Albumin Interaction
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Nb or serum albumin was coupled to CNBr-activated sepharose resin (GE Healthcare). For the in vitro pull-down assay, different concentrations of Nbs were incubated with the human serum albumin coupled resin. Samples were incubated for 15–30 min at 4°C with gentle agitation. The resin was collected and washed three times with a washing buffer and was boiled in the LDS sample loading buffer (Thermo) before SDS PAGE analysis.
6
LIN28A Binding Affinity Assay
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Biotin conjugated pre-let-7a miRNA (10 femtomoles) was tethered to Streptavidin-C1 MyOne magnetic beads. Beads were resuspended in 20mM Tris pH7.5, 50mM KCl, 5mM MgCl2, 1mM DTT, 5% Glycerol containing 100nM LIN28A-TAT (Peprotech) and a variable amount of competitor (let-7a, sponge construct, shRNA or polyA RNA; 5pM, 50pM, 500pM or 5nM). Beads were washed twice with 20mM Tris pH7.5, 50mM KCl, 5mM MgCl2 and 5% Glycerol and resuspended in 10ul of 1x LDS Sample loading buffer (Thermo) in RIPA. Bead resuspensions were boiled at 95°C for 5 minutes and profiled for the retention of LIN28A protein.
7
Immunoprecipitation of Med30 from Mouse Heart
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Heart tissue lysates from E10.5 Med30 cKO or control mouse were rotated overnight at 4°C in 300 μL of IP lysis buffer (Thermo Scientific, 87788) with antibody after taking 5% lysates use for input. 50 μL of PBS-washed protein G magnetic beads (Thermo Scientific,88847) were resuspended after pre-wash 3 times by 0.02% PBST and incubated in lysate–antibody complexes for 4 hours at 4°C. After washing 3 times by using IP lysis buffer, beads were incubated with 100 μL 50mM pH2.8 Glycine, 4× LDS Sample loading buffer (Thermo Scientific, NP0007) and 10× Sample reducing agent (Thermo Scientific, NP0004) incubate with at 70°C for 10 minutes. The immunoprecipitates and input lysate were gel electrophoresed and immunoblotted.
8
Phospho-Cyclin D3 Thr283 Analysis
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Pre-B cells were treated with FC162 (doses indicated in Figure 4 ) or vehicle (0.1% DMSO) for 3 h. Cells were then collected and lysed for 30 min on ice in TENT buffer (50 mM Tris, pH 8, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100) supplemented with 5 mM NaF, 2 mM NaVO3, 2 mM ß-glycerophosphate, 2 mM sodium pyrophosphate, and 1x complete protease inhibitor EDTA-free (Roche, Basel, Switzerland). Lysates were cleared by centrifugation for 10 min at 21,000× g at 4 °C. Protein lysates were denatured in LDS sample loading buffer (Life Technologies, Carlsbad, CA, USA) with 5% ß-mercaptoethanol at 95 °C for 5 min and electrophoresed on 4–12% Bis-Tris gradient gels (Life Technologies). Proteins were transferred to PVDF membranes and probed with primary antibodies for phospho-cyclin D3 Thr283 (ab55322, Abcam), total cyclin D3 (C-16, Santa Cruz Biotechnology, Inc, Dallas, TX, USA), and HSC-70 (B-6, Santa Cruz Biotechnology, Inc), and detected with HRP-conjugated secondary antibodies and ECL substrate (GE Healthcare, Marlborough, MA, USA). Immunoblots were performed in triplicate. Band densitometry values were calculated using ImageJ software.
9
Radioactive Labeling and SDS-PAGE Analysis
10
Protein Expression and Western Blotting
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Cells were lysed in TENT buffer (50 mM Tris, pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100) supplemented with 2 mM NaF, 2 mM NaVO3, 2 mM PMSF, and 1× cOmplete protease inhibitor cocktail (Roche) for 30 min on ice. Insoluble debris was pelleted by centrifugation at 21,000 g for 10 min at 4°C. Lysates were denatured in LDS sample loading buffer (Life Technologies) at 100°C for 5 min, and electrophoresed on 4–12% Bis-Tris gradient gels (Life Technologies). Proteins were transferred to PVDF membranes and probed with primary antibodies for: DYRK1A (7D10; Abnova), Cyclin D3 (C-16; Santa Cruz Biotechnology, Inc.), Cyclin D2 (M-20; Santa Cruz Biotechnology, Inc.), phospho-RB S807/811 (D20B12; Cell Signaling Technology), phospho-Src family Y461 (D49G4; Cell Signaling Technology), phospho-PDK1 S241 (C49H2; Cell Signaling Technology), Lyn (5G2, Cell Signaling Technology), phospho-NFATc2 S326 (sc-32994; Santa Cruz Biotechnology, Inc.), and FLAG (M2; Sigma-Aldrich) and detected with HRP-conjugated secondary antibodies and ECL substrate (GE Healthcare). β-Actin was detected using an HRP-conjugated primary antibody (C4; Santa Cruz Biotechnology, Inc.). Band densitometry values were calculated using ImageJ software.
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