A total of 1 × 106 of 4-week old BMMCs or one-day old DRG cultures were lysed in ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.3% Triton X-100, pH 8) with 1% of protease inhibitor cocktail III (Fisher Scientific). The insoluble debris was removed by spinning at 14,000 g for 20 min at 4°C. The protein concentration of each lysate was determined using Bradford protein assay (Sigma). For separation of CADM1 protein, 10% SDS resolving gel was used. After running the gel, the protein transfer was performed at 85 V for 90 min at 4°C (protein of interest 110 kDa). Then, the membrane was probed using rabbit anti-mouse CADM1 antibody (1:300, Santa Cruz) and mouse anti-GAPDH (1:5000, Thermo Fisher Scientific) and visualized using Li-cor system, Goat IRDye 800 anti-Rabbit 1:5000 and Goat IRDye 700 anti-Mouse 1:5000.
Protease inhibitor cocktail 3
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protease Inhibitor Cocktail III is a comprehensive solution designed to inhibit a broad range of proteases in biological samples. It contains a mixture of potent and specific inhibitors that target serine, cysteine, and metalloproteinases. This product is intended for use in research applications to preserve the integrity of proteins and prevent unwanted proteolysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Sivelestat, by Cayman Chemical (2 mentions)
Human gdna, by Promega (2 mentions)
Mouse anti gapdh, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Merck Group (1 mentions)
Mda elisa kit, by MyBioSource (1 mentions)
Gsh elisa kit, by MyBioSource (1 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Anti cd44, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail 2, by Thermo Fisher Scientific (1 mentions)
Vectastain abc kit, by Cell Signaling Technology (1 mentions)
Akt pan c67e7, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Mircury lna universal cdna synthesis kit, by Qiagen (1 mentions)
Mirna specific lna primers, by Qiagen (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Timstof pro, by Bruker (1 mentions)
11 protocols using protease inhibitor cocktail 3
1
CADM1 Protein Expression Analysis in BMMCs and DRG
2
Oxidative Stress and Inflammatory Biomarkers Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lysate from DRG was prepared by incubating the DRGs with 1% of protease inhibitor cocktail III (Fisher Scientific, St. Louis, MI, USA) for 1 h. The protein contents in the supernatant were quantified using a Bradford protein assay.
The oxidative stress parameters were measured using mice malondialdehyde (MDA) ELISA kit (MyBioSource MBS263626, San Diego, CA, USA), a mice glutathione (GSH) ELISA kit (MyBioSource MBS267424, San Diego, CA, USA), and a mice superoxide dismutase enzymes (SOD) ELISA kit (MyBioSource MBS034842, San Diego, CA, USA), following the manufacturer’s instructions.
The pro-inflammatory biomarkers were measured using a mouse nuclear factor KB ELISA Kit (MyBioSource MBS043224, San Diego, CA, USA) and the tumor necrosis factor kit assay (MyBioSource MBS825075, San Diego, CA, USA), following the manufacturer’s instructions.
The oxidative stress parameters were measured using mice malondialdehyde (MDA) ELISA kit (MyBioSource MBS263626, San Diego, CA, USA), a mice glutathione (GSH) ELISA kit (MyBioSource MBS267424, San Diego, CA, USA), and a mice superoxide dismutase enzymes (SOD) ELISA kit (MyBioSource MBS034842, San Diego, CA, USA), following the manufacturer’s instructions.
The pro-inflammatory biomarkers were measured using a mouse nuclear factor KB ELISA Kit (MyBioSource MBS043224, San Diego, CA, USA) and the tumor necrosis factor kit assay (MyBioSource MBS825075, San Diego, CA, USA), following the manufacturer’s instructions.
3
Protein Extraction and Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction from cells were isolated in RIPA lysis buffer (25 mM Tris-HCl (pH 7.4), 10% (v/v) glycerol, 150 mM NaCl, 2 mM EDTA, 1% (v/v) phosphatase inhibitor cocktail II (Thermo Scientific), and 1% (v/v) protease inhibitor cocktail III (Thermo Scientific)). Then protein concentrations was determined using a Thermo Scientific Pierce BCA Protein Assay Kit and denatured in 1× Laemmli’s gel loading buffer. Cellular protein was separated by SDS-PAGE and transferred to PVDF membranes, then probed with primary antibodies against SALL4, p-EGFR (Tyr1068), ERK1/2 (L34F12), p-ERK1/2 (Thr202/Tyr204), AKT (pan) (C67E7), p-AKT (Ser473), and β-actin (Cell Signaling). Signals were detected using a chemiluminescence kit (Thermo Scientific). The sections of human lung cancers were subjected to hematoxylin-eosin staining and immunohistochemical staining using a VECTASTAIN ABC Kit (rabbit/mouse IgG) with anti-SALL4 and anti-CD44 (Cell Signaling) antibodies.
4
Quantifying miRNA Transcripts via Ago2 Pulldown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SW1353 cells were transiently transfected with either precursor miR-140 or precursor miR-3085 for 48 h as above. Cell lysates were harvested with 0.5%(v/v) NP40, 150 mM KCl, 25 mM Tris–glycine pH7.5, 2 mM EDTA, 0.5 mM DTT, and 1 × protease inhibitor cocktail III (Thermo Fisher Scientific). Samples were pulled down with Ago2 antibody (MABE253, Sigma Aldrich) using Dynabeads Protein G (Thermo Fisher Scientific) and DynaMag Magnet at 4 °C, overnight. In order to quantify miRNA expression, targeted Ago2 was eluted with Cells-to-cDNA II lysis buffer (Thermo Fisher Scientific) and the miRCURY LNA Universal cDNA synthesis kit (Exiqon, Denmark) and miRNA-specific LNA primers (Exiqon, Denmark) for miR-140-3p, miR-29b-3p, and miR-3085-3p were used for quantification of mature miRNA transcripts by qRT-PCR.
5
Targeted Proteomic Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sequencing-grade modified trypsin was purchased from Promega (Fitchburg, WI). Iodoacetamide (IAA ), dithiothreitol (DTT ), triflfluoroacetic acid (TFA ), EDTA, urea, and tetraethylammonium borohydride (TEAB ) were obtained from Sigma (St. Louis, MO). Formic acid (FA ) was purchased from Buches (Germany). Protease inhibitor cocktail III, TMT kit, can, and pure water was purchased from Thermo Fisher Scientific (Waltham, MA). The 2-D Quant Kit was obtained from GE Healthcare (Buckinghamshire, UK). TIMS-TOF Pro (Bruker, Germany) was used for LC-MS/MS. The RNA inversion kit (dp431) was purchased from Tiangen company (China). All qRT-PCR kits were purchased from Ecoray Biological Company (Seoul, South Korea). The protein extraction kit, BCA protein quantitation kit, skimmed milk powder, polyvinylidene difluoride (PVDF ) membranes, SDS-PAGE gel preparation kit, PMSF, 10x protein loading buffer, Coomassie Brilliant Blue fast staining solution, developing, and fixing solution, ECL solution, and T protein marker were all purchased from Shanghai Wansheng Haotian Biotechnology Co. Ltd. (China). Goat anti-rabbit AMPD1 antibody (nbp2-24508; Novus Biologicals, Littleton, CO) and rabbit anti-mouse GAPDH antibody (sc-293335; Santa Cruz Biotechnology, Dallas, TX) were used for western blotting. The primers were synthesized by Shanghai Shenggong (China).
6
Extracellular Vesicle Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EVs were concentrated in Vivaspin 2, 5 kDa, concentrator columns to 20 µL and lysed using 10X RIPA lysis buffer containing protease inhibitor cocktail III (1:100; Thermo Fisher). Lysates from cells or EVs (20 µg) were loaded into a 4–12% Bis–Tris NUPAGE gel (Invitrogen) either under reducing (with DTT; GM130) or non-reducing (Alix, TSG101, CD9 and CD81) conditions and run at 125 V for 90 min in NUPAGE MES SDS buffer. Gels were subject to a wet transfer onto a nitrocellulose membrane using NUPAGE transfer buffer (15% methanol) at 150 V for 2 h at 4 °C. Membranes were blocked in 5% (w/v) milk in 0.1% (v/v) PBST (PBS Tween-20) for 1 h. Membranes were probed with antibodies against: TSG101 (Abcam; ab83, mouse, 1:500) Alix (Abcam; Ab117600, mouse, 1:2500), CD9 (Cambridge Biosciences / System Biosciences; EXOAB-CD9A-1; rabbit 1:1000), CD81 (Abcam; ab79559, rabbit, 1:1000), and GM130 (Abcam; ab52649, rabbit, 1:1000). The membranes were then washed three times with PBST then probed in milk buffer with either anti-Rabbit conjugated horseradish peroxidase (HRP) (Promega; w4011, 1:5,000) or anti-mouse HRP conjugated secondary antibody (Promega; w402B, 1:20,000). The membranes were washed again three times with PBST, then developed using ECL reagent (Amersham; Pico) for 5 min (RT) and imaged using a Bio-Rad Gel Doc XR +.
7
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown on 10-cm plates until fully confluent, placed on ice, washed with ice-cold PBS, and lysed with ice-cold Triton X-100 lysis buffer (150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris, pH 7.4, and 1% Triton X-100) containing 2× protease (Protease Inhibitor Cocktail III, RPI) and phosphatase inhibitors (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific). Six 10-cm plates were used per IP. Before cell lysis, 7 µg of antibody or IgG control (011-000-003; Jackson ImmunoResearch Laboratories) was incubated with 50 μl Protein G Dynabeads (Invitrogen) in 200 µl PBS with 0.02% Tween-20 for 10–12 h at 4°C with constant end-to-end rotation. For the AGO2 IP, 8 µg of antibody or IgG isotype control (ab18443; Abcam) were used. After washing the antibody-conjugated beads three times with immunoprecipitation (IP) lysis buffer, cell lysates were incubated with the bead-conjugated antibodies 12 h at 4°C with constant end-to-end rotation. Beads were then washed three times with IP lysis buffer and eluted using 50 mM dithiothreitol (DTT; Sigma-Aldrich) and 0.5% sodium dodecyl sulfate (SDS) in lysis buffer at 58°C for 30 min, with constant agitation. Eluted proteins were separated by SDS–PAGE and analyzed by immunoblotting as described below.
8
Molecular Pathways Modulation in Osteoarthritis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SW1353 cells were plated in 6-well plate wells at 1.5 × 105 cells/well and left to adhere overnight. Cells were transiently transfected with 50 nM miR-3085-3p mimic (Qiagen), siRNA (Qiagen) or non-targeting controls (Qiagen) for 48 h. After serum starvation for another 24 h, cells were stimulated with IL-1β (5 ng/ml) (First Link (UK) Ltd) for 30 min or TGFβ1 (4 ng/ml) (R&D Systems) for 2 h and washed twice in ice-cold phosphate buffered saline (PBS). Whole cell lysates were harvested into ice cold RIPA buffer (50 mM Tris–HCL pH7.6, 150 mM NaCl, 1% (v/v) Triton x-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10 mM NaF, 2 mM Na3VO4, 1 × protease inhibitor cocktail III (Thermo Fisher Scientific)). Samples were separated on reducing SDS-PAGE, transferred to PVDF membrane and probed overnight at 4 °C. The p65 (#8242), phospho-p65 (#3033), IκBα (#4814), phosphor-IκBα (#2859),SMADs (#3103, #9513, #38,454), phospho-SMADs (#9523), MyD88 (#4283), β-catenin (#9582), phospho-β-catenin (#9561) and GAPDH (#2118) (all from Cell Signaling Technology, used at recommended concentrations) were detected using HRP-conjugated secondary antibodies (DAKO), visualised using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific), and imaged by ChemiDoc MP Imaging System (Biorad)13 (link).
9
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or tissue samples were lysed in radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate] plus 1X protease inhibitor cocktail III (Alfa aesar) with 0.1% SDS (cells) or 0.3% SDS (tissues). Protein concentrations were measured by using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Ten micrograms (cells) or 50 μg (tissues) of whole protein lysate was mixed with sample buffer, boiled for 10 min, and loaded on 10% SDS-PAGE gels followed by electrophoresis. Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore) and then probed with antibodies against MMP9, MMP3, GAPDH, Nrf2, HO-1, or β-actin in blocking buffer. HRP-conjugated goat anti-rabbit IgG or anti-mouse IgG antibodies (BD Biosciences) were used as secondary antibodies. The membranes were then incubated with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) followed by the detection of protein signals by using X-ray films. The quantification of protein signal was measured by using ImageJ.
10
Investigating NET-Mediated Epithelial Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NETs were pre-incubated with A1AT protein from human plasma (Cayman Chemical #24560) 1h before exposure to HBE. Individual components of NETs were incubated with wtCFBE41o- to assess the effect of each on cytotoxicity and monolayer integrity. These included 5µg/ml human gDNA (Promega), 500ng/ml citrullinated histones (Cayman Chemical #501620), 100mU/ml human NE protein (Athens Research & Technology #16-14-051200, ART), 100µU/ml human PR3 protein (ART #16-14-161820) and 4.5mU/ml human CG protein (ART #16-14-030107). These were compared to media control, and 5µg/ml NETs. The concentration of individual NET components was chosen to reflect that found in our isolated human NETs. In epithelial cell-free experiments, NETs were pre-incubated with protease inhibitors: human A1AT (Cayman Chemical 24560), sivelestat (Cayman Chemical #177749), and cathepsin G inhibitor I (Cayman Chemical #14928) all at 1µg/ml and 100µg/ml, and protease inhibitor cocktail III at 0.1µM and 100µM (Alfa Aesar #J64283) (1h at 37°C) and assayed for protease activity (43 (link)).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!