1 × 106 cells were harvested and lysed in RIPA buffer containing 1x Protease Inhibitor Cocktail, 1 mM PMSF, 5 mM NaVO4 and 5 mM NaF. Extracts were loaded into 4-15% Mini-Protean TGX SDS Page gels (Bio-Rad). Proteins were transferred to PVDF membranes. Membranes were blocked in 5% milk/PBS-T buffer for 30 min and incubated either overnight at 4°C or 1 hour at room temperature with the following antibodies: 4EBP1 (total or pThr37/46), S6K1 (total or pThr389), Akt (total or pSer473), mTor (total or pSer2448) (Cell Signaling Technology), Gapdh (Millipore) and anti-rabbit/mouse secondary antibodies. Membranes were incubated with ECL or ECL Plus reagents and exposed to X-ray films (Thermo Fisher Scientific).
Mini protean tgx sds page gels
Manufactured by Bio-Rad
Sourced in United States
The Mini-Protean TGX SDS Page gels are pre-cast polyacrylamide gels designed for the separation and analysis of proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. These gels provide consistent and reliable separation of proteins based on their molecular weight.
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Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
X ray film, by Thermo Fisher Scientific (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Gapdh, by Merck Group (2 mentions)
4e bp1, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Mammalian pe lb buffer, by GoldBio (2 mentions)
Y1175 phospho vegfr 2, by Cell Signaling Technology (2 mentions)
Qiashredder, by Qiagen (1 mentions)
Chemidoc gel imaging system, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Goat anti rabbit igg h l hrp, by Thermo Fisher Scientific (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Lane marker reducing sample buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Tsg 101 antibody, by Thermo Fisher Scientific (1 mentions)
15 protocols using mini protean tgx sds page gels
1
Quantitative Immunoblotting of mTOR Pathway
2
Whole Cell Extract Protein Analysis
3
Western Blot Protein Analysis Protocol
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Protein samples were prepared by adding 2× Laemmli sample buffer (Bio-Rad, 1610737) to cell pellets followed by passing through QIA shredder (QIAGEN, 79656). Denatured proteins were separated by 4%–15% Mini-Protean TGX SDS-PAGE gels (BioRad, 4561096) and transferred to PVDF membranes. Membranes were blocked with 5% milk in the TBST buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% tween-20) followed by incubation with primary antibodies at 4 °C overnight. Membranes were washed 3 times with TBST buffer and then incubated with anti-mouse/rabbit secondary antibodies conjugated to HRP (Jackson) for 30 min at room temperature. After 3 times TBST buffer wash, proteins on the PVDF membrane were detected by chemiluminescence using Clarity™ Western ECL Substrate (Biorad, 1705060) and a ChemiDoc™ Gel Imaging System according to manufacturer’s instructions.
4
CDK7 Kinase Activity Assay
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Activity assays were performed in 25 mM Hepes-KOH pH 7.7, 150 mM KCl, 5 mM MgCl2, 5 mM β-mercaptoethanol, 5% glycerol. The 100-µl -reactions containing 500 nM CAK, 500 µM (YSPTSPS)3KKKK-biotin substrate peptide (synthesized by GenScript), and 2 mM ATP were incubated for 1, 2, 3, and 5 h at 30 °C. Assays for mass spectrometric analysis of phosphopeptides (SI Appendix, Fig. S6F ) were performed under identical conditions, except that the concentration of substrate peptides was lowered to 20 µM.
Proteins and peptides were separated on 4 to 20% MiniProtean TGX SDS/PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes using the TransBlot Turbo Western blotting system (Bio-Rad). The membrane was transferred into TBST (50 mM Tris⋅HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) with 5% bovine serum albumin and incubated for 2 h, followed by incubation with primary antibodies (rat α-CTD-phospho-Ser5 IgG, clone 3E8, EMD Millipore and rabbit α-CDK7 IgG, clone C-4, Santa Cruz Biotechnology) at 4 °C for 16 h. After washing with TBST, the membrane was incubated with secondary antibodies conjugated to fluorescent dyes (α-rabbit Cy5 and α-rat Cy3) for 1.5 h at room temperature. The fluorescent signal was recorded using a ChemiDoc imaging system (Bio-Rad).
Proteins and peptides were separated on 4 to 20% MiniProtean TGX SDS/PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes using the TransBlot Turbo Western blotting system (Bio-Rad). The membrane was transferred into TBST (50 mM Tris⋅HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) with 5% bovine serum albumin and incubated for 2 h, followed by incubation with primary antibodies (rat α-CTD-phospho-Ser5 IgG, clone 3E8, EMD Millipore and rabbit α-CDK7 IgG, clone C-4, Santa Cruz Biotechnology) at 4 °C for 16 h. After washing with TBST, the membrane was incubated with secondary antibodies conjugated to fluorescent dyes (α-rabbit Cy5 and α-rat Cy3) for 1.5 h at room temperature. The fluorescent signal was recorded using a ChemiDoc imaging system (Bio-Rad).
5
Detection of Exosomal TGF-β Protein
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For detection of exosomal TGF-β, 20 µg of exosomes in 40 µl PBS were lysed using Lane Marker Reducing Sample Buffer (ThermoFisher Scientific), heated at 95°C for 5 min and separated using 12% Mini-PROTEAN® TGX™ SDS/PAGE Gels (BioRad, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes using the Transblot Turbo System (BioRad). Membranes were blocked with 5% BSA/TBS-T for 1 h at room temperature and incubated with TGF-ß antibody (RRID : AB_2063354, Cell Signaling Technology, Danvers, MA, USA) or TSG101 antibody (RRID : AB_2548734, ThermoFisher Scientific) over night at 4°C. The next day, membranes were washed three times with TBS-T and incubated with Goat anti-Rabbit IgG (H+L), HRP (RRID : AB_228341, Thermo Fisher Scientific) for 40 min at room temperature. Membranes were developed and analyzed with the Chemi Doc MP Imaging System and Image Lab 6.0.1 software (Biorad).
6
Western Blot Protein Detection Protocol
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Cytoplasmic extracts were diluted to 1× Laemmli sample buffer, heated at 95°C for 5 min, and electrophoresed on Bio-Rad Mini-PROTEAN TGX SDS-PAGE gels at 150V in 1× Tris/glycine buffer containing 1% SDS (w/v). Proteins were then transferred to an Immobilon-FL PVDF membrane (Millipore Sigma IPFL00010) at 4°C and at 100V for 60 min in 1× Tris/glycine buffer containing 20% methanol (v/v) and 0.1% SDS (w/v). Membranes were blocked at room temperature in 3% BSA (w/v) in TBS for at least 30 min. Primary antibody staining was performed with rabbit anti-RNMT antibody (Proteintech 13743-1-AP, 1:500 dilution) or rabbit anti-EEF2 (One World Lab; 1:500 dilution) in 3% BSA (w/v) in TBS. Following three 10-min washes with TBS-T, membranes were incubated in the dark for 30 min in 3% BSA (w/v) in TBS containing a 1:10,000 dilution of anti-rabbit Alexa Fluor 680 (Thermo Fisher A21109). Membranes were washed with TBS-T as before, and Western blots were visualized on a Li-Cor Odyssey at 700 nm.
7
Western Blot Analysis of Cellular Proteins
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Whole-cell and chromatin extract we prepared according to methods described previously(39 (link), 40 (link)). Denatured samples were separated on 4–15% Mini-Protean TGX SDS-PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes by wet transfer using Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Bio-Rad). Membranes were blocked in 5% milk/TBS-T and incubated with indicated primary antibodies overnight at 4 °C. HRP-conjugated anti-mouse/rabbit secondary antibodies, were incubated for 1h at room temperature. Proteins were detected by ECL (Pierce) or Clarity Max (Bio-Rad). Quantification of bands was performed using ImageJ. See Table S7 for antibodies and dilutions.
8
Quantitative Immunoblotting of mTOR Pathway
9
Western Blot Analysis of Cell Cycle
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Denatured samples were separated on 4-15% Mini-Protean TGX SDS-PAGE gels (Bio-rad).
Protein was transferred to methanol-activated PVDF membranes (Bio-rad) by wet transfer (1x Pierce Transfer Buffer, 10% methanol) or using high molecular weight transfer conditions for the Bio-rad TransBlot Turbo (Bio-rad). Membranes were blocked in 5% milk/TBS-T and incubated with indicated primary antibodies for 1.5h at room temperature or overnight at 4˚C. Membranes were then washed and incubated with HRP-conjugated anti-mouse/rabbit secondary antibodies (Jackson Labs) for 1h at room temperature. Proteins were detected by ECL (Pierce) or Clarity (Bio-rad) detection reagents and exposure to X-ray film (Pierce).
For analysis of cell cycle, FUCCI reporter ES cells 33 were collected by trypsinization and sorted on a FACS AriaII (BD Biosciences) into mCherry+ (G0/G1) and BFP+ (S/G2/M) cell fractions.
Cells were pelleted and lysed in RIPA buffer and clarified by centrifugation for 10 min, 13,000 g at 4˚C. Lysates from the same number of cells were used for western blotting.
Protein was transferred to methanol-activated PVDF membranes (Bio-rad) by wet transfer (1x Pierce Transfer Buffer, 10% methanol) or using high molecular weight transfer conditions for the Bio-rad TransBlot Turbo (Bio-rad). Membranes were blocked in 5% milk/TBS-T and incubated with indicated primary antibodies for 1.5h at room temperature or overnight at 4˚C. Membranes were then washed and incubated with HRP-conjugated anti-mouse/rabbit secondary antibodies (Jackson Labs) for 1h at room temperature. Proteins were detected by ECL (Pierce) or Clarity (Bio-rad) detection reagents and exposure to X-ray film (Pierce).
For analysis of cell cycle, FUCCI reporter ES cells 33 were collected by trypsinization and sorted on a FACS AriaII (BD Biosciences) into mCherry+ (G0/G1) and BFP+ (S/G2/M) cell fractions.
Cells were pelleted and lysed in RIPA buffer and clarified by centrifugation for 10 min, 13,000 g at 4˚C. Lysates from the same number of cells were used for western blotting.
10
Quantifying ACE2, TMPRSS2 Protein Levels
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Protein from 5 × 105 cells was harvested by directly lysing cells in 2x Laemmli Sample Buffer (BioRad Laboratories, Hercules, CA, USA) +/- 5% 2-mercaptoethanol. Lysed cells were boiled for 5 min at 95 °C and spun at maximum speed to pellet insoluble debris. Supernatants were run on 7% or 10% SDS-PAGE Mini-Protean TGX gels (BioRad) at 200 V for 30-45 min. Proteins were transferred to 0.22 μm nitrocellulose membrane (ThermoFisher). Membranes were blocked with 5% milk, immunoblotted with 1:1000 dilution of primary antibody, washed, incubated with 1:2500 dilution of hrp conjugated secondary antibodies, washed, and developed using SuperSignal Picoluminescent substrate (ThermoFisher) and a BioRad ChemiDoc Touch. Primary antibodies used were goat anti-hACE2 (R&D AF933); rabbit anti-TMPRSS2 (Abcam ab109131, Cambridge, UK); mouse anti-GAPDH (ThermoFisher AM4300).
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