Cell culture dishes at 80% confluence were washed with ice-cold PBS1X, lysed with 500 µl of lysis buffer (50 mM Tris, pH 7,2, 350 mM NaCl, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, 10 mM MgCl2, protease inhibitor cocktail complete tablets (Roche)) and centrifuged for 5 min at 13,000 RPM at 4°C. The supernatant was incubated with bacterially produced glutathione-S-transferase (GST)-PAK-CD fusion protein, containing the RAC and Cdc42-binding region from human PAK1B (Sander et al., 1998 (link)) bound to glutathione-coupled Sepharose beads at 4°C for 45 min. The beads and proteins bound to the fusion protein were washed three times with a wash buffer (50 mM Tris, pH7.2, 1% Triton X-100, 150 mM NaCl, 20 mM MgCl2, protease inhibitor cocktail complete tablets (Roche)) and eluted in Laemmli sample buffer (60 mM Tris pH 6,8, 2% SDS, 10% glycerine, 0,1% bromophenol blue) and then analysed for bound RAC1 molecules by western blot using a RAC1 antibody.
Protease inhibitor cocktail tablets complete
Manufactured by Roche
Sourced in Germany
The Protease Inhibitor Cocktail Tablets Complete is a laboratory product designed to inhibit the activity of proteases, a class of enzymes that break down proteins. The tablets provide a comprehensive solution to prevent protein degradation during various experimental procedures.
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Lab products found in correlation
Superase in, by Thermo Fisher Scientific (2 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Super signal west dura extended duration subtract kit, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Rat ngf, by R&D Systems (1 mentions)
Phosphatase inhibitor cocktail tablets phosstop, by Roche (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (1 mentions)
Thrombin, by Merck Group (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Anti runx2, by Santa Cruz Biotechnology (1 mentions)
Aminocaproic acid, by Merck Group (1 mentions)
Bovine fibrinogen, by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
12 protocols using protease inhibitor cocktail tablets complete
1
Rac1 Activation Assay Protocol
2
Monitoring NGF/BDNF Signaling in PC12 Cells
3
Osteogenic Differentiation of Mesenchymal Cells
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Bovine fibrinogen, thrombin, aminocaproic acid, L-Ascorbic acid and glycerol 2-phosphate were purchased from Sigma (St. Louis, MO). Rat tail type I collagen was purchased from BD Bioscience (San Jose, CA). Fetal bovine serum (FBS), HEPES buffer solution, and penicillin-streptomycin solution were from GibcoBRL (Carlsbad, CA), and ascorbic acid-free α-MEM was from WelGene (Daegu, Korea). The MicroBCA assay kit was from Pierce-Thermo (Rockford, IL). Qunat-iT PicoGreen dsDNA-assay kit was from Invitrogen (Eugene, OR). West-Zol was from Intron Biotechnology (Seoul, Korea). A Dual-GloTM Luciferase Assay System was from Promega (Madison, WI). Protease inhibitor cocktail tablets (Complete) were from Roche (Basel, Switzerland). Anti-Runx2, anti-actin, and HRP-conjugated IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-fibronectin and anti-vitronectin antibodies were from Chemicon (Temecula, CA).
4
Quantitative Protein Analysis of Heart Tissue
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Heart tissues (20-30 mg), collected NRCMs, or CFs were lysed in RIPA buffer (Servicebio, G2002) supplemented with Protease Inhibitor Cocktail Tablets complete (Roche) and Phosphatase Inhibitor Cocktail Tablets Pho-STOP(Roche). BCA Protein Assay Kit (Thermo) was used to quantify the extracted total protein from heart tissue or cells. Total protein (30 μg) was separated by SDS-PAGE electrophoresis followed by transferring to polyvinylidene fluoride (PVDF) membranes. Following blockade with TBS containing 5% skim milk, membranes were incubated with primary antibodies overnight at 4°C. The next day, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Abbkine). ChemiDoc™ Imaging System (Bio-Rad) was used to analysis the blots. Immunoblots were quantified using Image Lab™ software (version 6.0). These following primary antibodies were used in this study: GAPDH (CST, 2118), Phospho-STAT3 (Tyr705) (CST, 9138), STAT3(CST, 9139), Histone H3 (ABCAM, ab5176), p-SMAD2 (Ser465/467) (CST, 3180), SMAD2 (CST, 3130), p-SMAD1/5(Ser463/465) (CST, 9516), SMAD1/5 (SANTA, sc6201), p-SMAD3 (Ser423/S425) (ABCAM,ab52903), SMAD3 (CST, 9513), BAX (CST, 2772), BCL2 (CST, 2870), TGF-β (Abcam, ab66403), FN (SANTA, sc-6952), α-SMA (ABCAM, ab5694), ANP (SANTA, sc-515701), β-MHC (SANTA, sc-530090), and BNP (ABCAM, ab239510).
5
Mitochondrial Dynamics Assay Protocol
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Fetal calf serum, penicillin/streptomycin, and basal Eagle's medium were purchased from GIBCO, Invitrogen (Carlsbad, CA, USA). Dihydroethidium (DHE), MitoTracker green, and MitoTracker red CMH2XRos were purchased from Molecular Probes, Invitrogen (Carlsbad, CA, USA). Poly-l-lysine, trypsin, trypsin inhibitor, DNAse, cytosine arabinoside, DMSO (dimethyl sulfoxide), staurosporine, MitoTEMPO, and reagents for polyacrylamide gel electrophoresis (PAGE) were acquired from Sigma (St. Louis, MO, USA). Protease inhibitor cocktail tablets (Complete) were purchased from Roche (Mannheim, Germany), and phosphatase inhibitor minitablets were obtained from Thermo Scientific (Rockford, USA). ProSieve Quad Color Protein Marker was purchased from Lonza (Rockland, Maine, USA). Polyvinylidene fluoride (PVDF) membranes and Immobilon Western HRP substrate were acquired from Millipore (Concord Road, Billerica, MA, USA). Antibodies against Drp1, Drp1 (Ser616), and GAPDH were from Cell Signaling Technology (Danvers, MA, USA); peroxidase-conjugated anti-mouse was purchased from Jackson ImmunoResearch (West Grove, PA, USA).
6
Linc-MAF-4 Transcript Abundance Analysis
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In vitro differentiated TH1 cells were UV-crosslinked at 400 mJ/cm2 in ice-cold D-PBS and then pelleted at 1350g for 5 min. The pellet was resuspended in ice-cold lysis buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40) supplemented with 0.5 mM β-mercaptoethanol, Protease Inhibitor Cocktail Tablets cOmplete, EDTA-free (Roche) and SUPERase•In (Ambion) and left rocking at 4 °C until lysis was complete. Debris was centrifuged at 13000g for 10 min. The lysate was precleared with Dynabeads® Protein G (Novex®) for 30 min at 4 °C and then incubated for 2 h with 7 μg of antibodies specific for EZH2 (Active Motif - 39875); LSD1 (Abcam – ab17721), or HA (Santa Cruz - sc7392) as mock control. The lysate was coupled with Dynabeads® Protein G (Novex®) for 1 h at 4 °C. Immunoprecipitates were washed for five times with lysis buffer. RNA was then extracted following mirVana miRNA Isolation Kit (Ambion) protocol. The RNA transcript abundance of Linc-MAF-4 or of the negative controls β-actin, RNU2.1 and a region upstream the TSS of linc-MAF-4 (linc-MAF-4 control) was assessed by RT-qPCR.
7
Detecting Polyubiquitinated TAK1 and TRAF6
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For protein-protein interaction analysis, HEK293T cells were washed twice with ice-cold 1xphosphate-buffered saline, and lysed in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 3% Glycerol, 1% NP-40, protease inhibitor cocktail tablets complete, EDTA-free(Roche)). The cell lysates were sonicated and centrifuged at 13,000 × g for 10 min at 4°C. To detect the polyubiquitinated form of TAK1 and TRAF6, in vivo ubiquitination assay were performed as follows. Cells were lysed in 0.4 ml TNET buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton-X-100). Supernatants were denatured in 1% SDS by boiling for 15 min to remove noncovalently attached proteins, and then diluted to 0.1% SDS with regular TENT buffer. Then, supernatants were incubated with indicated antibody for 3 h at 4°C and rotated with Protein A-agarose (Millipore) for 3 h or overnight (for endogenous protein immunoprecipitation) at 4°C. After six times washes with lysis buffer, the immunoprecipitated materials were boiled in 40 μl 2 × SDS loading buffer and subjected to Western blotting.
8
Purification of Retroviral-Like Particles
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HepG2 or Vero2-2 cells were transduced (MOI 2) with the indicated HSV-1 amplicon vector and RVLPs were harvested 48 hpt as described previously [48 (link),49 ]. Briefly, cells were scraped into the medium and cell membranes were disrupted by repeated cycles of thawing/freezing. The cell debris was removed by centrifugation at 1400× g and filtration through a 0.45 µm filter. The cleared supernatant was loaded onto a 10% sucrose cushion and concentrated at 100,000× g for 2 h at 16 °C. For protection, protease inhibitor (protease inhibitor cocktail tablets complete, mini, ethylenediaminetetraacetic acid (EDTA)-free, 1 tablet per 10 mL, Roche Diagnostics, Mannheim, Germany) was added to the supernatant.
9
Linc-MAF-4 Transcript Abundance Analysis
10
Western Blot Analysis of c-Myc Protein
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Transfected Bm5 and High Five cells were collected and lysed by sonication (Branson) in a lysis buffer consisting of Tris-Cl 50 mM, NaCl 150 mM, EDTA 1 mM, Triton-X-100 1%, Sodium Dodecyl Sulphate (SDS) 0.5% and Protease Inhibitor Cocktail Tablets Complete (Roche). Next, the total amount of proteins was quantified by means of a Bicinchoninic acid assay, after which 18 μg of each sample were separated by SDS-polyacrylamide gel electrophoresis. Then, the proteins were transferred to a Trans-Blot Turbo Mini PVDF membrane using the Trans-Blot Turb Blotting System (Bio-Rad). The blots were washed and blocked with a skimmed powder milk solution 5% for 2 hours. Anti-c-Myc antibodies produced in rabbit (Sigma-Aldrich) were diluted (1:5000) and incubated with the blots overnight at room temperature. Washing was then performed, followed by 2 hours of incubation with Polyclonal Goat Anti-Rabbit Immunoglobulins/HRP (Dako), diluted 1:50000. Finally, the blots were washed and the detection was performed with Super Signal West Dura Extended Duration Subtract Kit (Thermo Scientific). The chemiluminescent bands were visualized using a ChemiDoc™ MP Imaging System with Image Lab Software (Bio-Rad).
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