Monolayer-cultured hiPSC-CMs were rinsed 2x with 1x PBS and lysed with Pierce RIPA lysis buffer (Thermo Fisher, 89901) supplemented with Halt protease and phosphatase inhibitor (Invitrogen, 78443) and dithiothreitol (Roche, 3483-12-3). Lysates were rocked at 4C for 20 min and centrifuged 10 min at 15,000g, with supernatant collected. Total protein concentration of supernatant was assessed by Bradford assay (Bio Rad, 5000006) with 560 nm absorbance per manufacturer’s protocol using BSA standards (Thermo Fisher, 23208). Lysates were diluted with 4x Laemmli SDS Sample Buffer (Bio Rad, 1610747) to 1 μg/μL total protein concentration.
Laemmli sds sample buffer
Manufactured by Bio-Rad
Sourced in United States
Laemmli SDS sample buffer is a preformulated solution for preparing protein samples for SDS-PAGE analysis. It contains key components such as SDS, reducing agent, and tracking dye. The buffer helps denature and solubilize proteins prior to electrophoretic separation.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay, by Bio-Rad (2 mentions)
Bsa standard, by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Pierce ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
Superblock tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Mab374, by Merck Group (1 mentions)
Genetools software, by Syngene (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Stealth rna, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Immunotools (1 mentions)
Supersignal west pico plus substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Dithiothreitol (dtt), by Roche (1 mentions)
Nhs peg4 biotin, by Thermo Fisher Scientific (1 mentions)
High capacity neutravidin agarose resin, by Thermo Fisher Scientific (1 mentions)
Gfp trap agarose beads, by Proteintech (1 mentions)
15 protocols using laemmli sds sample buffer
1
Protein Extraction from hiPSC-CMs
2
Western Blot Analysis of LDLR Expression
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Liver samples were homogenized in RIPA buffer containing Complete Protease and PhosStop Phosphatase Inhibitor Cocktail (Roche). Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples (20 μg protein) in 4x Laemmli SDS Sample Buffer (Bio-Rad) were loaded on Criterion™ TGX™ Tris-Glycine 4–20% precast gels (Bio-Rad). Proteins were separated in SDS-PAGE and transferred to nitrocellulose membrane (Trans-Blot® Turbo™ Transfer System, Bio-Rad). Membrane was blocked (SuperBlock TBS Blocking buffer, Thermo Fisher Scientific) for 30 mins at room temperature and incubated with primary antibodies (anti-LDLR: Abcam, #ab180623, 1:2000; anti-GAPDH: Millipore, MAB374, 1:10000) in the blocking buffer overnight at 4 °C with gentle agitation. Next day, the membrane was incubated with secondary antibodies (anti-rabbit IgG: 1:2000, #7074; anti-mouse IgG – 1:10000, #7076, Cell Signaling Technology) for 1 hour at room temperature in TBS buffer containing 2.5% milk and 0.1% Tween 20. Signal was developed by enhanced chemiluminescence detection method (Supersignal West Dura, Thermo Fisher Scientific) and captured with G:BOX mini imaging system (Syngene, Frederick, MD, USA). Band densities were analyzed by GeneTools software (Syngene), and GAPDH was used as a loading control.
3
Th2 Differentiation and STAT6 Knockdown
4
Enzymatic Deglycosylation of Proteins
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Cleavage of the N-glycan was performed using PNGase Fand Endo H as described [4 (link)]. Briefly, total cell lysates and biotinylated surface proteins were denatured in denaturing buffer at 95 °C for 10 min. The protein mixtures were incubated with PNGase F (500 units) or Endo H (1000 units) for 18 hr at 37 °C. The protein was boiled for 10 min at 95 °C subjected to Western blot analysis after adding an equal volume of 2× laemmli SDS sample buffer (BioRad).
5
Crosslinking Protein Complexes for SDS-PAGE
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Protein eluates from SEC were crosslinked at 2.5 μM concentration with 250 μM dithiobis succinimidyl propionate (DSP) (Thermo Scientific) at room temperature for 30 min. The reactions were quenched with 50 mM final concentration of Tris-HCl pH 7.5 for 15 min. Aliquot from each reaction was mixed with 2x Laemmli SDS sample buffer (Bio-Rad) for SDS-PAGE analysis.
6
LyzC-EGFP Secretion Assay Protocol
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5 × 105 cells stably expressing LyzC-EGFP were plated in a 6-well plate for the secretion assay. After 24 h, cells were pretreated for 30 min with 70 µM 2-APB and DMSO. After that, cells were incubated in a growth medium containing 70 µM 2-APB and DMSO for 1 h. Cells and media were collected separately. Cells were lysed using RIPA buffer. The collected growth media was incubated overnight with GFP-trap beads at 4°C. The following day, the beads were washed 4 times with DPBS, and then protein was eluted from the beads by boiling it in 2X Laemmli SDS sample buffer (Bio-Rad). Cell lysates and IP fractions were analyzed by Western blotting.
7
Protein Expression Analysis of NR4A3 and MSANTD3
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Tumor protein lysates were extracted using RIPA buffer containing protease inhibitor (no. 0505648900; Roche Applied Science, 200 Victoria Dr Gilroy, Gilroy, CA, USA) and phosphatase inhibitor (no. 04906837001; Roche Applied Science) cocktails. Laemmli SDS sample buffer (Bio-Rad, 2000 Alfred Nobel Drive, Hercules, CA, USA) was used for protein denaturation. Protein samples were loaded into SDS page gel and transferred onto nitrocellulose membranes. Membranes were incubated with primary antibodies; anti-NR4A3 (NGFI-B gamma/NOR-1, clone # H7833, mouse, R&D systems, 614 McKinley Place NE, Minneapolis, MN, USA), anti-MSANTD3 (#PA5-66513, rabbit, Thermo Fisher) and anti-ACTB (#AC-15, mouse; Sigma-Aldrich, PO Box 14508, St. Louis, MO, USA). After incubation with secondary antibody, bands were detected using SuperSignal West Pico PLUS substrate (# 34577, Thermo Fisher).
8
Proteomic analysis of hiPSC-CMs
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Monolayer-cultured hiPSC-CMs were rinsed twice with 1× PBS and lysed with Pierce RIPA lysis buffer (Thermo Fisher, 89901, Waltham, MA, USA) supplemented with Halt protease and phosphatase inhibitor (Invitrogen, 78443, Waltham, MA, USA) and dithiothreitol (Roche, 3483-12-3, Basel, Switzerland). Lysates were rocked at 4 °C for 20 min and centrifuged 10 min at 15,000× g, with supernatant collected. Total protein concentration of supernatant was assessed through a Bradford assay (Bio Rad, 5000006, Hercules, CA, USA) with 560 nm absorbance per manufacturer’s protocol using BSA standards (Thermo Fisher, 23208, Waltham, MA, USA). Lysates were diluted with 4× Laemmli SDS Sample Buffer (Bio Rad, 1610747, Hercules, CA, USA) to 1 μg/μL total protein concentration.
9
Immunoblot Analysis of Extracellular Vesicles
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Isolated vesicles were either lysed in RIPA buffer/inhibitor cocktail (ThermoFisher) or solubilized directly in Laemmli SDS sample buffer (BioRad). RIPA-mediated lysis occurred for 20 min on ice, with occasional vortex, followed by centrifugation at 20,000 × g for 20 min to pellet cellular debris. Protein concentration of the supernatant was determined by the BCA method (Pierce). Samples were resolved by Tris/glycine PAGE, transferred to PVDF membranes (0.2 μm pore), washed (TBS/0.1% Tween 20 (TBS/T20)), blocked (TBS/T20/5% non-fat dry milk), and incubated with primary antibody overnight (in blocking buffer). The membranes were then incubated with streptavidin HRP (in blocking buffer) if required, washed, and developed by enhanced chemiluminescence. Antibodies and reagents utilized for immunoblot analysis included: from Southern Biotech, anti-IgM HRP, anti-IgG2a HRP; from BD, anti-CD9 biotin (clone KMC8), anti-CD81 biotin (clone Eat2), control purified mouse IgM; from Pierce, streptavidin HRP.
10
Biotinylation and Affinity Purification
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Cells, seeded at 8 × 105 per well of 6-well plates and grown for 4 days (primary kidney) or for 24 hr after transfection (HEK293T), were washed 3 times with ice-cold PBS and treated with 0.4 mM NHS-PEG4-Biotin (Thermo, Waltham, MA) for 30 min on ice. The biotinylation reaction was quenched and excess reagents were washed out using PBS containing 0.1 M glycine. Cells were then lysed in 700 μl of cell lysis buffer. Three hundred μl of the cell lysate were incubated with 5 μl of High Capacity NeutrAvidin agarose resin (Thermo) for 2 h at 4°C with rotation. The resins were washed 3 times with cell lysis buffer and boiled in 2× Laemmli SDS sample buffer (Bio-Rad, Hercules, CA) supplemented with 0.1 M DTT.
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