A2780, HEC1B and HeLa cells were plated and treated according to the following conditions and as described above for the cell viability or the clonogenic assays. Cells were washed with cold PBS and lysed 1% SDS, 150 mM NaCl, 5 mM NaF, 1% Tween 20, 0.5% NP40, 50 mM Tris–HCl pH 7.5 and 1× Halt protease and phosphatase inhibitor (Pierce). Equivalent amounts of protein lysates were resolved via 4–15% mini-PROTEIN TGX gels (Bio-Rad) and transferred to PVDF membranes. Membranes were blocked for 1 h using 5% non-fat dry milk (Bio-Rad) and incubated with primary antibody overnight at 4 °C, secondary antibody for 4 h at ambient temperature or overnight at 4 °C, and SuperSignal West Dura Chemiluminescent Substrate (ThermoFisher Scientific) for 5 min. Anti-pATM (S1981) and anti-β actin were from Abcam. Anti-ATM was from Thermo Scientific. Anti-ATR, anti-pChk1 (S345), anti-Chk1, anti-pChk2 (Thr68), anti-Chk2, anti-PARP, anti-cleaved PARP, anti-caspase 3, anti-cleaved caspase 3, goat anti-mouse and goat anti-rabbit IgG HRP-linked were from Cell Signaling Technologies. All primary antibodies were used at a 1:1000 dilution. Immunoblots were first probed for cleaved PARP followed by total PARP (Fig. 5B ). Similarly, blots were first probed for cleaved caspase 3 followed by total caspase 3 (Fig. 5C and D ). Images were acquired using a ChemiDoc XRS+ system (Bio-Rad).
Mini protein tgx gel
Manufactured by Bio-Rad
Sourced in United States, Japan, Germany, United Kingdom
The Mini-Protein TGX gels are precast polyacrylamide gels designed for the rapid and efficient separation of proteins. These gels provide consistent and reliable performance in protein electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Ecl solution, by GE Healthcare (4 mentions)
Laemmli sample buffer, by Bio-Rad (4 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Trans blot turbo system, by Bio-Rad (3 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Protein assay method, by Bio-Rad (3 mentions)
Skim milk, by Bio-Rad (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (3 mentions)
Amersham ecl western blotting, by Cytiva (3 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Pierce detergent compatible bradford assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Anti gadph, by Cell Signaling Technology (2 mentions)
Orthovanadate, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
46 protocols using mini protein tgx gel
1
Immunoblotting Analysis of DNA Damage Response
2
Western Blot Analysis of NMP4 Protein
3
Extracellular Vesicle Protein Profiling
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Extracellular vesicles from shSCR and shSDC2 hMSCs were lysed in 1× RIPA buffer (Cell Signaling, Danvers, MA, USA) and 1× mini protease inhibitor cocktail (cOmplete™). After adding Laemmli's SDS Sample Buffer (6×; Boston BioProducts, Ashland, MA, USA), the lysed EVs were boiled at 100 °C for 5 min, and then, equal protein concentration was electrophoresed on 4–20% Mini Protein TGX Gels (Bio‐Rad Laboratories, Hercules, CA, USA). Antibodies for blotting (SDC2, ALIX, Tsg101, syntenin‐1, Histone H3, CD63, CD81, CD9, and β‐actin) are detailed in Table 1 . Protein expression was assessed using imagej software.
4
Tyro3 Protein Isolation and Western Blot
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Protein isolation from cells was performed using RIPA lysis buffer containing 1x protease inhibitor cocktail Complete (Roche, Basel, Switzerland), 1 mM PMSF, and 1 mM orthovanadate (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was determined using DC™ Protein Assay (Bio-Rad, Feldkirchen, Germany). Western blots were performed using precast Mini Protein TGX gels and the semi-dry Trans-Blot TurboTM System (Bio-Rad, Germany). Antibodies and related secondary antibodies (DAKO, Tokyo, Japan) were used at a dilution of 1:1000 in TBST for Anti-Tyro3 (Cell Signalling, Danvers, MA, USA, #5585). Anti-GADPH (Cell Signalling, USA, #5174) was used as a loading control.
5
Western Blot Protein Detection
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Cells were lysed using RIPA buffer with complete protease inhibitor (Roche), 1mM Na3VO4 and 1mM PMSF. Lysates were quantified with the Pierce BCA Protein Assay Kit (ThermoFisher) and electrophoresed on MINI-protein TGX gels (BioRad) and transferred onto PVDF membrane (BioRad). Membranes were immunoblotted with indicated antibodies. All primary antibodies were diluted in 1% milk or BSA in TBST and were incubated overnight at 4°C. Blots were incubated in secondary antibody for 1 hour at room temperature, also in 1% milk or BSA.
6
Western Blot Analysis of Epigenetic and Metabolic Proteins
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Cells were lysed with NP40 buffer at a pH of 7.6 containing 50 mM Tris, 150 mM NaCl, 1% NP40, 0.2% Lauryl Maltoside, 1 mM Sodium orthovanadate (Sigma-Aldrich, St. Louis, MO, USA), and 1 × protease inhibitor cocktail of cOmplete™ (Roche, Basel, Switzerland). Protein concentration was determined using a DC™ Protein Assay kit (Bio-Rad, Hercules, CA, USA), and proteins were immunoblotted as described previously [56 (link)]. Then, 15 µg denatured protein was separated on precast Mini Protein TGX gels (Bio-Rad) and transferred to a nitrocellulose membrane using the semi-dry Trans-Blot Turbo™ system. Antibodies and related secondary antibodies (Dako / Agilent, Santa Clara, CA, USA) were used at a dilution of 1:1,000 in TBST for Anti-EZH2 (Cell Signaling Technology, Danvers, MA, USA, #5246), Anti-H3K27me3 (ActiveMotif, Carlsbad, CA, USA, #39155), and Anti-MTHFD2 (Abnova, Heidelberg, Germany, #H00010797-M01). Anti-PARK7 (Abcam, Camebridge, UK, #ab18257) and Anti-GADPH (Cell Signaling Technology, #5174) were used as loading controls.
7
Western Blot Analysis of AKAP12
8
Quantifying AID Protein Expression
9
Quantifying AID Protein Expression
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Whole cell extracts were obtained from purified mouse B cells or cell lines treated with 1 μM PI3K inhibitors using GST-FISH buffer (10 mM MgCl2, 150 mM NaCl, 1% NP-40, 2% Glycerol, 1 mM EDTA, 25 mM HEPES (pH 7.5)) supplemented with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na3VO4. Extracts were cleared by centrifugation at 12,000 r.p.m. for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 12% Mini-PROTEIN TGX gels (BIO-RAD), transferred on nitrocellulose membrane (GE Healthcare), blocked with 5% Skim milk (BIO-RAD). Primary antibodies for immunoblotting included: rat monoclonal anti-mouse AID (mAID-2 clone, eBioScience, CAT NO #14-5959-82), mouse monoclonal anti-human AID (ZA001, Life Technologies, CAT NO # 39-2500), rabbit monoclonal anti-PI3K p110δ (Y387, Abcam, CAT NO #32401), rabbit polyclonal anti-β–actin (Sigma, CAT NO #A2066), rabbit monoclonal anti-Phospho-AKT (S473) (D9E, Cell Signaling Technology, CAT NO #4060), rabbit monoclonal anti-AKT (pan) (C67E7, Cell Signaling Technology, CAT NO #4691). Membranes were developed with ECL solution (GE Healthcare). AID protein abundance was measured by ImageJ software and normalized for the β-actin intensity of the corresponding lane.
10
Nepenthes Pitcher Urease Extraction and Analysis
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Proteins were extracted from Nepenthes leaf or pitcher tissue using 50 mM phosphate buffer (pH 7.5) containing 2% PVPP, 50 mM NaCl, 1 mM EDTA and 20 mM DTT (DTT was added fresh before extraction). Protein separation by SDS-PAGE and blotting were performed using Miniprotein TGX gels (Bio-Rad) and Trans blot turbo blotting system (Bio-Rad) respectively. Polyclonal anti-jackbean (Thermo Fisher Scientific), anti-A. thaliana urease, and anti-A. thaliana UreD antibodies17 were used for immunoblot urease detection and indicated in the particular experiments. Transient expression of NhUrease in Nicotiana benthamiana was performed according to16 (link), 17 . For functional tests, NhUrease and accessary proteins from A. thaliana, UreD, UreF, and UreG, were coexpressed. Urease activity was measured as described48 (link).
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