Cell lysates were obtained in lysis buffer containing Halt Protease & Phosphatase Single-Use Inhibitor Cocktail (Thermo Scientific), 20 µg of protein were loaded on 12% polyacrylamide gel and then transferred on nitrocellulose membrane. The membrane was blocked with Odyssey blocking solution (LI-COR, Lincoln, Nebraska) at room temperature for 1 h and followed by an incubation overnight with primary antibodies p53 DO-1 mouse monoclonal IgG (1:5,000, Santa Cruz Biotechnology) and Cdk2 rabbit polyclonal IgG (1:10,000, Santa Cruz Biotechnology) at 4 °C. The membrane was then incubated with secondary antibodies Goat anti-mouse IgG (1:5,000, IRDye® 680RD, LI-COR) and Goat anti-rabbit (1:10,000, IRDye® 800CW, LI-COR) at room temperature for 1 h in dark. Odyssey detection system (CLx, LI-COR) was used to visualize the immunoreactive bands. Image J software was used to quantify the density of the bands.
Halt protease phosphatase single use inhibitor cocktail
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Halt Protease & Phosphatase Single-Use Inhibitor Cocktail is a ready-to-use solution that contains a mixture of protease and phosphatase inhibitors. It is designed to help preserve the integrity of protein and phosphoprotein samples during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking solution, by LI COR (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Sodium fluoride, by Merck Group (1 mentions)
Ab52818, by Abcam (1 mentions)
Ab49900, by Abcam (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Sequencing grade trypsin, by Merck Group (1 mentions)
Protease inhibitor tablet, by Merck Group (1 mentions)
Lc ms grade formic acid, by Thermo Fisher Scientific (1 mentions)
12 protocols using halt protease phosphatase single use inhibitor cocktail
1
Western Blot Analysis of p53 and Cdk2
2
Nanoparticle-Induced Cellular Signaling Pathway Analysis
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The nanoparticle treated cells were rinsed with PBS and lysed in p300 lysis buffer (250 mM NaCl, 0.1% NP-40, 20 mM sodium phosphate, 30 mM sodium pyrophosphate, 5 mM EDTA, and 10 mM sodium fluoride, adjusted at pH 7.0) supplemented with Halt protease & phosphatase single-use inhibitor cocktail (Thermo Fisher Scientific). Whole cell lysate (10–30 μg) was separated by polyacrylamide gel electrophoresis (10, 12, and 15%) and electrotransferred to PVDF membrane Hybond-P (GE Health Sciences, Piscataway, NJ). After blocking in in 1× TBST with 5% nonfat dry milk, blots were incubated with specific primary antibodies overnight at 4 °C and visualized by horseradish peroxidase-conjugated second antibody using Pierce ECL Western blotting substrate (Thermo Fisher Scientific). Antibodies were purchased from Cell Signaling (Danvers, MA), LC3, p62, pERK1/2, ERK1/2, pJNK, pP38, and Rab5, and from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA), actin, JNK, p38, Ubiquitin, Flotilin, Caveolin-1.
3
Immunoblotting Analysis of Cell Signaling
4
Quantitative Immunoblot Analysis of LDLR
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HepG2 cells were directly lysed in RIPA buffer with proteases the Halt Protease & phosphatase Single-use Inhibitor cocktail (ThermoFisher, Waltham, MA, USA). After centrifugation for 5 min at 12,000× g, the supernatant was stored at −20 °C. Protein quantification was performed with the Protein Assay Dye Reagent (Bio-Rad, Hercules, CA, USA). Then, 40 μg of protein were denatured with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) during 5 min at 95 °C, loaded onto mini-PROTEAN TGX 4–20% of SDS-PAGE gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Primary antibodies anti-LDL receptor [EP1553Y] (1:1000, ab52818, Abcam, Cambridge, UK) and anti-beta actin [AC-15] (HRP) (1:20.000, ab49900, Abcam, Cambridge, UK) were used for protein detection. Chemiluminescence signals were detected using Supersignal West Pico Plus (ThermoFisher, Waltham, MA, USA) in a BOX Chemi XRQ (SYNGENE, Cambridge, UK) following the manufacturer’s instructions. Quantitative immunoblots were analyzed with ImageJ software (Version 1.46r, National Institutes of Health, Bethesda, MD, USA) [45 (link)]. LDLR levels were quantified and normalized using β-actin as an internal control.
5
Antibody Formulations Analysis Protocol
6
Nanoparticle Pulldown from MC3T3-E1 Cells
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MC3T3-E1 cells were incubated with NP1-MNP or NP1-MNP-PEG for 72 h. Cells were rinsed with cold PBS once and lysed in E1A lysis buffer (250 mM NaCl, 0.1% NP-40, and 50 mM HEPES, adjusted at pH 7.5) supplemented with Halt protease & phosphatase single-use inhibitor cocktail (Thermo Fisher Scientific). Nanoparticles were “pulled-down” from the lysate using Dynal magnets (Dynal Biotech ASA, Oslo Norway) ∼8 h initially followed by washing with lysis buffer. Two subsequent pulldowns were performed at 4 °C to further purify sample (Figure 5 A).
7
Protein Extraction from Brain Tissue
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Samples from human brain were stored at − 80 °C and ground with a mortar in a frozen environment with liquid nitrogen to prevent thawing of the samples resulting in tissue powder. Mouse brains were quickly dissected on an ice-cold plate and the different brain structures stored at − 80 °C. Protein extracts were prepared by homogenizing brain structures in ice-cold extraction buffer [250 mM sucrose, 20 mM Tris–HCl (pH 7.4), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA)] containing cocktail of protease and phosphatase inhibitors (Halt Protease & Phosphatase Inhibitor Single-Use Cocktail; ThermoFisher) using 20 strokes with a Teflon-coated pestle. Homogenates were centrifuged at 3000 rpm for 5 min at 4 °C. The resulting supernatant was collected, and protein content determined by BCA (Pierce, Rockford, IL, USA).
To prepare cell lysates for Western blotting neurons were collected in 1X Laemmli sample buffer (150 ml/well on 24 well plates; Bio-Rad Laboratories). Cells were lysed and boiled for 5 min. A volume of 15 ml was subsequently used for electrophoresis.
To prepare cell lysates for Western blotting neurons were collected in 1X Laemmli sample buffer (150 ml/well on 24 well plates; Bio-Rad Laboratories). Cells were lysed and boiled for 5 min. A volume of 15 ml was subsequently used for electrophoresis.
8
Protein Isolation and Detection in Kidney Cortex
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The kidney cortex was lysed with a buffer containing cell lysis buffer (Cell Signaling Technology), EDTA, and Halt Protease & Phosphatase inhibitor single-use cocktail (Thermo Fisher Scientific). Samples with an equivalent amount of protein were analyzed by SDS-PAGE. Proteins were electroblotted onto PVDF membranes (Bio-Rad laboratories) and membranes were blocked with PVDF Blocking Reagent (TOYOBO, Tokyo, Japan) then incubated sequentially with primary and secondary antibodies. Protein detection was performed using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) and LAS-4000 (Fujifilm, Tokyo, Japan).
9
Quantifying NAD+ and ART1 Interaction
10
Western Blot Analysis of Cell Signaling Proteins
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Protein lysates were prepared from T-cell pellets using RIPA (Sigma-Aldrich) solution and the Halt™ Protease/Phosphatase Inhibitor Single-Use Cocktail (Thermo Fisher Scientific). Protein concentrations were determined using a BCA protein assay Kit (Thermo Fisher Scientific). Proteins (>10 µg) were separated on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) system and transferred by wet blotting (Biorad, Hercules, California, USA). Western blots were probed using primary antibodies against Tubulin, CPT1a, Bcl-2, Bcl-xL, and Mcl-1 (Cell Signaling Technology Inc., Danvers, Massachusetts, USA and Supplementary Table 3 ). Bands were detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgGs (Cell Signaling Technology; Agilent) and an ECL substrate (Cell Signaling Technology Inc.).
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