EVs (1.4 × 1010 particles) and cell lysates (20 μg of total proteins) were separated in polyacrylamide gel (TGX FastCast Acrylamide Kit, 7.5%, Bio-Rad, #1610171) (TGX FastCast Acrylamide Kit, 12%, Bio-Rad, #1610175) electrophoresis and transferred onto a PVDF membrane (Millipore, #IPVH00010). The antibodies to EV markers: Alix (Sigma-Aldrich, #SAB4200477), TSG101 (Abcam, #ab30871), and CD63 (BD Bioscience, #556019), and antibody to Golgi marker: Golgi Reassembly Stacking Protein 1 (GORASP1) (Sigma-Aldrich, #SAB4100063) were used to detect separated proteins. The membranes were then incubated with anti-rabbit IgG-HRP (Cell Signaling Technology, #7074) or anti-mouse IgG-HRP (Cell Signaling Technology, #7076). Binding of specific antibodies was detected with Western chemiluminescent HRP substrate (Millipore, #WBKLS0500), visualized, and quantified with FUSION SOLO S (Vilber Lourmat). The intensity level of the band corresponding to each molecule was normalized by its intensity in the corresponding cell lysate.
Tgx fastcast acrylamide kit
Manufactured by Bio-Rad
Sourced in United States
The TGX FastCast Acrylamide Kit is a pre-mixed solution for preparing polyacrylamide gels for electrophoresis. It contains the necessary components to cast gels quickly and conveniently.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Las 4000 mini analyzer, by Cytiva (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
A6154, by Merck Group (1 mentions)
Hatf00010, by Merck Group (1 mentions)
C digit, by LI COR (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Image studio, by LI COR (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using tgx fastcast acrylamide kit
1
Extracellular Vesicle Protein Analysis
2
Immunoblotting Protein Extraction and Separation
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Proteins were extracted from each sample with SDS loading buffer (3% SDS, 15% glycerol, 180 mM Tris-HCl, 4% β-mercaptoethanol). For subcellular fractionation samples, protein concentrations were determined by the BCA protein assay (Pierce, 23250, Thermo Scientific), and equal quantities of proteins were subjected to SDS-PAGE. For immunoisolation samples, equal volumes of SDS loading buffer were added to magnetic isolated beads. Samples were incubated in 75°C for 20 min to better immunoblot transmembrane proteins. Briefly, samples were loaded to 10% SDS-PAGE gels (TGX FastCast Acrylamide Kits, 1610183, Bio-Rad) and transferred to 0.45 mm NC membranes (HATF00010, Millipore) for 2 hr. Membranes were subjected to 5% milk in TBST buffer (Tri-buffered Saline with Tween 20) at room temperature for 1 hr to block non-specific binding. Primary antibodies were prepared in TBST and incubated with membranes at 4°C overnight. Secondary antibodies were either anti-rabbit IgG HRP (A6154, Sigma, 1:5000 dilution) or anti-mouse IgG HRP (715-035-150, Jackson ImmunoResearch, 1:5000 dilution). Final results were visualized using chemiluminescence (Merck Millipore) in e-BLOT Touch Imager (e-BLOT Life Science, Shanghai).
3
Western Blot Protein Analysis Protocol
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Collected cell pellets were suspended in lysis buffer (20 mM Tris-HCl at pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM DTT, plus a protease inhibitor cocktail [Roche 11873580001]) and then incubated for 5 min on ice. Chromatin pellets were collected by centrifugation at 20,400g for 5 min at 4°C and then suspended in SDS sampling buffer (50 mM Tris-HCl at pH 6.8, 2% SDS, 0.01% bromophenol blue, 10% glycerol, 5% 2-mercaptoethanol) for boiling at 95°C. Protein samples were resolved with 12% TGX FastCast acrylamide kits (Bio-Rad 1610175) and then transferred to PVDF membranes (Amersham 10600057). Membranes were blocked using 5% skim milk (BD 232100) in PBST (0.1% Tween 20 in phosphate-buffered saline [PBS]) for 30 min and then incubated with primary antibodies (Supplemental Table S3 ) diluted in 5% skim milk in PBST. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Supplemental Table S3 ), and then chemiluminometric signals were detected using ECL (Amersham RPN2232) and C-Digit (Li-cor 3600). Acquired images were processed using Image Studio (Li-cor) and ImageJ software (https://imagej.net/Welcome ).
4
Protein Extraction and Western Blotting Protocol
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Protein extraction and Western blotting were performed as described previously.14 (link) The protein content was quantified using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific), and protein samples (20 mg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) using a gel prepared from the TGX Fastcast Acrylamide kit (10%, Bio‐Rad) according to the manufacturer's protocol. Block Ace (KAC Co., Ltd) was used for membrane blocking. Band signals were detected by the luminescent Image Analyzer LAS‐4000 mini (Fujifilm). The original full images of membranes are shown in Figures [Link] , [Link] , [Link] .
5
Immunoprecipitation and Western Blotting
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Antibodies used in this study are reported in Supplementary Table S3 . Reagents and chemicals include: Ionomycin calcium salt (Sigma # I3909), Protein G-Agarose (Sigma #11243233001), Streptavidin Sepharose High Performance bead (Sigma #17-5113-01), Calmodulin Sepharose 4B (Sigma #17-0529-01), Dynabeads™ Protein G for Immunoprecipitation (Thermo Fisher Scientific #10004D), jetPRIME®, DNA and siRNA transfection reagent, Polyplus-transfection® reagent (VWR #89129-922), TRIzol™ Reagent (Thermo Fisher Scientific #15596018), Quantitect reverse transcriptase (Qiagen #205313), Xpert Protease inhibitor cocktail (GenDEPOT #P3100-100), Xpert Phosphatase inhibitor cocktail (GenDEPOT #P3200-020), Penicillin-Streptomycin (GenDEPOT #CA005-100), 10× PBS Buffer (GenDEPOT #P2100-100), DMEM, High Glucose with L-Glutamine (GenDEPOT #CM002-050), Opti-MEM™ Reduced Serum Medium (Thermo Fisher Scientific #31985070), FBS Opti-Gold, Us Origin (GenDEPOT #F0900-050), iTaq Universal SYBR® Green Supermix (Bio-Rad #1725124), Immun-Blot PVDF Membrane (Bio-Rad #1620177), TGX™ FastCast™ Acrylamide Kit (Bio-Rad #161-0173, #161-0175), Precision Plus Protein™ Dual Color Standards (Bio-Rad #1610394), Blotto, non-fat dry milk (Santa-Cruz # sc-2325), SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific #34076), VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vectorlabs #H-1500).
6
Western Blot Quantification of GFP and DsRed
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Extracted TSP (10 µg) from both PCPs and Agrobacterium cells was loaded on 10% (w/v) sodium dodecyl sulphate–polyacrylamide gel (TGX™ FastCast™ Acrylamide Kit, Bio-Rad, USA), separated by electrophoresis, and transferred onto a nitrocellulose membrane (Whatman® Protran®, Sigma Aldrich). To specifically detect the presence of GFP and DsRed, the western blot was probed with the rabbit polyclonal anti-GFP antibody (ExBio, Czech Republic) at 1:1,000 dilution, and the rabbit polyclonal anti-RFP antibody (MBL International, USA) at 1:1,000 dilution. The secondary antibody used for the detection of both proteins was alkaline phosphatase-conjugated goat anti-rabbit IgG (H + L) (Sigma Aldrich) at 1:30,000 dilution. The blots were developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate tablets (Sigma Aldrich). The band intensities were quantified with the ImageJ software (Version 1.48). GFP (Abcam, United Kingdom) and DsRed (Fraunhofer IME, Germany) proteins in the range of 0.005–1.0 mg/mL were used to create the standard curves.
7
Western Blot Protein Quantification
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Whole-cell lysates were prepared using 1X Lysis Buffer (Cell Signaling Technology). Samples were separated by SDS-PAGE using either EZ-Run Gel Solution (Thermo Fisher Scientific) or TGX Fast Cast Acrylamide Kit (BioRad, Hercules, CA, USA) gels and transferred to nitrocellulose membranes (GE Healthcare Life Sciences). The membranes were blocked and probed as previously described (Perron and Dodd, 2009 (link)). The blots were developed using either the SuperSignal West Pico (Thermo Fisher Scientific) or Trident Pico Western HRP (GeneTex, Irvine, CA, USA) chemiluminescent substrates using Blue X-ray film (Phenix Research Products, Candler, NC, USA) or the Omega Lum G Imaging System (Gel Company, San Francisco, CA, USA). Densitometric analysis was performed using ImageJ 1.49v software.
8
Western Blot Analysis of Cell Signaling
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The cells were lysed in lysis buffer, and 20 µL of protein lysate sample was fractionated on polyacrylamide gels (TGX™ FastCast™ Acrylamide Kit; Bio-Rad Laboratories, Hercules, CA, USA) and then electroblotted to nitrocellulose membranes. The membranes were blocked with 5% skim milk in phosphate buffered saline-Tween 20 (PBS-T). The membranes were incubated with primary and then secondary antibodies. They were then treated with enhanced chemiluminescence detection reagents (Amersham™; Cytiva, Marlborough, MA, USA), and chemiluminescent signals were visualized as bands using a LAS 4000 mini analyzer (Cytiva).
Antibodies against phospho-cdc2 (Try15), cyclin D1, cyclin B1, cleaved caspase-3, caspase-3, phospho-AKT (Ser473), AKT, phospho-WNK1 (Thr60), WNK1, phospho-GSK-3β (Ser9), GSK-3β, phospho-mTOR (Ser2448), and mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Monoclonal beta-actin antibody (FUJIFILM Wako Pure Chemical Corp.) was used to probe an internal control.
Antibodies against phospho-cdc2 (Try15), cyclin D1, cyclin B1, cleaved caspase-3, caspase-3, phospho-AKT (Ser473), AKT, phospho-WNK1 (Thr60), WNK1, phospho-GSK-3β (Ser9), GSK-3β, phospho-mTOR (Ser2448), and mTOR were purchased from Cell Signaling Technology (Beverly, MA, USA). Monoclonal beta-actin antibody (FUJIFILM Wako Pure Chemical Corp.) was used to probe an internal control.
9
Western Blot Protein Analysis
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Cells were dissolved in lysis buffer, and 10 µL of each protein sample was fractionated on polyacrylamide gels (TGX™ FastCast™ Acrylamide Kit; Bio-Rad Laboratories, Hercules, CA, USA) and then electroblotted onto nitrocellulose membranes. The membranes were blocked and incubated with primary antibodies against β-actin (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) or KLF4 (Novus Biologicals, Littleton, CO, USA), and then the membranes were incubated with horseradish peroxidase-conjugated secondary antibody. The membranes were then treated with enhanced chemiluminescence detection reagents (Amersham™; Cytiva, Marlborough, MA, USA), and chemiluminescent signals were visualized as bands using a LAS 4000 mini analyzer (Cytiva).
10
Western Blot Analysis of Apoptosis Markers
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The lysis buffer was RIPA buffer with protease inhibitor cocktail. The cells were added to lysis buffer and then underwent an alternant vortex and stored at 4 °C for half an hour. Then the cell lysis was centrifuged at 16,000 × g for 20 min at 4 °C. The Laemmli buffer was used to denature the samples. TGX™ FastCast™ Acrylamide Kit (Bio-rad), APS, and TEMED were used to make the gels. The PVDF membrane was blocked by 5% BSA solution for 1 h. Primary (first) antibodies included Caspase-3 antibody (Cell Signaling Technology, #9662), anti-fatty acid synthase antibody (Abcam, ab22759), Erk1/2 (Thr202/Tyr204) antibody (Cell Signaling Technology, #9101S), and anti-Bcl-xL antibody [E18] (Abcam, ab32370). The membrane was incubated with the primary antibody overnight at 4 °C, rinsed with TBST, and then incubated with the second antibody for 1 h at room temperature. ECL Prime western blotting reagents (GE Healthcare) were used to develop. The developer was myECL Imager (Thermo Fisher Scientific).
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