To extract protein, ahRPE were washed and then lysed with RIPA buffer containing phosphatase and protease inhibitor cocktails (Roche) on ice. Samples were scraped on ice, agitated for 30 min at 4 °C and then centrifuged at 12,000 rpm for 20 min at 4 °C to remove debris. The supernatant was collected, aliquoted to prevent freeze-thaw cycles, and protein concentration was subsequently determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts were separated by BoltTM 4–12% Bis-Tris Plus Gels (Thermo Fisher Scientific) and transferred onto iBlot® 2 PVDF Mini Stacks (Thermo Fisher Scientific) membranes and were probed with antibodies (Supplementary Table 4 ). Proteins of interest were detected with Peroxidase AffiniPure Goat Anti-Mouse IgG, F(ab’)2 Fragment Specific antibody (1:10000, Jackson ImmunoResearch, Cat #:115–035–072) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) antibody (1:10000, Jackson ImmunoResearch, Cat #:111–035–045), and subsequently visualized with ECLTM Prime Western Blotting Detection Reagent (GE Healthcare) according to the provided protocol. All samples were stored at −80 °C. Uncropped western blots for Figs. 3d, 5c, d , and 6d can be found in Supplementary Figs 5 and 6 .
Iblot2 pvdf mini stacks
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBlot2 PVDF Mini Stacks is a lab equipment product designed for protein transfer during Western blotting experiments. It provides a compact and efficient solution for the transfer of proteins from polyacrylamide gels to PVDF membranes. The product includes all the necessary components for the transfer process.
Automatically generated - may contain errors
Lab products found in correlation
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecltm prime western blotting detection reagent, by GE Healthcare (1 mentions)
Peroxidase affinipure goat anti rabbit igg h l antibody, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Rabbit anti mouse igg hrp, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Skim milk, by Fujifilm (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Ccd camera, by Vilber (1 mentions)
Novex tris glycine sds sample buffer 2, by Thermo Fisher Scientific (1 mentions)
Novex wedgewell 4 12 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Sc 7294, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Ecl peroxidase labeled anti mouse antibody, by GE Healthcare (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
9 protocols using iblot2 pvdf mini stacks
1
Protein Extraction and Western Blotting Protocol
2
SARS-CoV-2 Spike Protein Expression in Cell Lines
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All vaccine candidates were transformed at 25 multiplicity of infection (MOI) into THP-1, A549, and RD cell lines seeded each 2.5 × 105 cells. Western blot analysis was performed after harvested supernatant and lysate of cells transformed with vaccine candidates to verify the accumulation of S protein secretion. Following boiling in sample buffer without β-ME for detection of the S protein trimer, the protein samples were resolved on 4–12% Bis-tris gels. To prevent saturating the signal of spike antigen overexpression, each sample was exposed for less than 15 s.
Cells were lysed in RIPA buffer containing protease inhibitors (Merck, Kenilworth, NJ, USA), and the culture medium was concentrated using 50,000 MWCO microcon (Merck, Kenilworth, NJ, USA). Antibody-antigen complexes on PVDF membranes (iBlot 2 PVDF Mini Stacks, Thermo Fisher Scientific, Waltham, MA, USA) were quantified using Quantity One 1-D analysis software (Bio-Rad, Hercules, CA, USA). The following antibodies were used: rabbit anti-SARS-CoV-2 spike antibody (GeneTex, Irvine, MA, USA), mouse anti-β-actin antibody (Merck, Kenilworth, NJ, USA), goat anti-rabbit IgG H&L (HRP) (Abcam, Cambridge, MA, USA), and rabbit anti-mouse IgG (HRP) (Merck, Kenilworth, NJ, USA).
Cells were lysed in RIPA buffer containing protease inhibitors (Merck, Kenilworth, NJ, USA), and the culture medium was concentrated using 50,000 MWCO microcon (Merck, Kenilworth, NJ, USA). Antibody-antigen complexes on PVDF membranes (iBlot 2 PVDF Mini Stacks, Thermo Fisher Scientific, Waltham, MA, USA) were quantified using Quantity One 1-D analysis software (Bio-Rad, Hercules, CA, USA). The following antibodies were used: rabbit anti-SARS-CoV-2 spike antibody (GeneTex, Irvine, MA, USA), mouse anti-β-actin antibody (Merck, Kenilworth, NJ, USA), goat anti-rabbit IgG H&L (HRP) (Abcam, Cambridge, MA, USA), and rabbit anti-mouse IgG (HRP) (Merck, Kenilworth, NJ, USA).
3
Western Blot Analysis of Nfatc1 in Murine Cells
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Cultured murine cells were lysed in RIPA buffer (Nacalai Tesque, Kyoto, Japan). Protein concentration was determined using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). The denatured lysate mixed with Novex™ Tris–Glycine SDS Sample Buffer (2×; Thermo Fisher Scientific) was loaded onto Novex™ WedgeWell™ 4–12% Tris–Glycine gels (Thermo Fisher Scientific) for electrophoresis. The gels were electroblotted onto a PVDF membrane using iBlot 2 PVDF mini stacks (Thermo Fisher Scientific). Membranes were blocked with Blocking One (Nacalai Tesque) for Nfatc1 detection and with 5% skim milk (FUJIFILM Wako Pure Chemical Corporation) for β-actin. The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at room temperature for 1 h. ECL Prime Western Blotting Detection Reagent (GE Healthcare Bioscience, Chicago, IL, USA) was added to the membranes, and the chemiluminescent signal was detected using a CCD camera (Vilber, Collégien, France). The primary antibodies used were mouse monoclonal antibodies against Nfatc1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7294, 1:1000 dilution) and β-actin (Sigma-Aldrich, A1978, 1:2000 dilution). The secondary antibody was an ECL peroxidase-labeled anti-mouse antibody (GE Healthcare Bioscience. 1:10,000 dilution).
4
Western Blot Analysis of Membrane Proteins
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Cells were lysed with Cell Lysis Buffer (Cell Signaling Technology) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). For Western blot to confirm expression of membrane-localized CD4 fusion protein in Fig. 5B , cells were lysed in RIPA buffer made using recipe found here: https://www.abcam.com/protocols/general-western-blot-protocol#Solutions%20and%20reagents . Lysates were mixed with NuPAGE LDS Sample Buffer (Life Technologies) and NuPAGE Sample Reducing Agent (Life Technologies) and were run on Bolt 4–12% Bis-Tris Plus gels (Invitrogen). Gels were transferred with iBlot2 PVDF Mini Stacks (Invitrogen) and iBlot2 (Invitrogen). Blots were probed with antibodies for mCherry (GTX128508, GeneTex), GFP (sc-99G, Santa Cruz Biotechnology), Tid1 (EPR12414, abcam or RS-11, Santa Cruz Biotechnology), p53 (DO-1, Santa Cruz Biotechnology), CHOP (9C8, Novus Biologicals), ATF6-N (70B1413.1, Novus Biologicals), pEIF2a (9721, Cell Signaling Technology), EIF2a (9722, Cell Signaling Technology), Bax (6A7, Santa Cruz Biotechnology), MDM2 (sc-965, Santa Cruz Biotechnology), Cyclophillin D (E11AE12BD4, abcam), Hsp90 (C45G5, Cell Signaling Technology), Cox IV (3E11, Cell Signaling Technology), and GAPDH (D4C6R, Cell Signaling Technology). Blots were imaged using Bio-Rad ChemiDoc MP Imaging System or Azure Biosystems Sapphire Biomolecular Imager.
5
Histone Acetylation Analysis by Western Blot
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Isolated histones were separated on a 4–20% SDS-PAGE gel and transferred to a PVDF membrane (iBlot® 2 PVDF Mini stacks, Invitrogen) using the iBlot® 2 dry blotting system on program P0 for 7 min. Membranes were blocked in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing 5% milk powder and probed for H3K9ac (Abcam ab61231; 1:5000) or H4ac (K5 + K8 + K12 + K16 acetyl; Abcam ab10807; 1:5000) primary antibodies diluted in TBS-T. This was followed by anti-rabbit HRP (Abcam ab97095; 1:5000) secondary antibody and visualisation using Pierce SuperSignal® West Pico chemiluminescent substrate.
6
Virus Purification and Western Blot
7
Western Blotting of Organoid Proteins
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Western blotting was performed as described previously57 (link). In brief, organoids were collected and lysed in PierceTM RIPA Buffer (ThermoFisher Scientific, Cat. #89900), and a cocktail of protease and phosphatase inhibitors (Roche). Organoids were then sonicated, and protein concentration was quantified using PierceTM bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific, Cat. #23227) according to the manufacturer’s instructions. Samples were then heated for 10 min at 100 °C before loading. An equal amount of proteins was loaded and separated using Mini-PROTEAN TGX Stain-Free-Gels (Bio-Rad, Cat. #4568044), separated proteins were then transferred onto iBlot 2 PVDF Mini Stacks (Invitrogen, Cat. #IB24002). The membrane was then blocked with 5% Skim Milk in TBS-T (20 mM Tris–HCl, pH 7.6, 136 mM NaCl, and 0.1% Tween-20) for 1 h at RT, followed by primary antibody incubation diluted in 5% BSA for 12 h at 4 °C. The primary antibodies used in this experiment are listed in Supplementary Table 5 . The membrane was then washed three times with 1× TBST for 10 min each at RT before incubation with secondary antibody diluted 1:5000 in 5% Skim Milk in 1× TBST for 1 h at RT. The membrane was washed again three times with 1× TBST for 10 min each at RT before visualization with Clarity Western ECL Substrate (Bio-Rad, Cat. #170-5060).
8
Cas9 and Stomatin Protein Detection
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Protein concentrations were determined by Bradford assay as described previously (61 (link)). Protein extracts of equal amounts were separated by SDS-PAGE (NuPAGE 4 to 12% [wt/vol] bis-Tris protein gels; Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (iBlot2 PVDF ministacks; Invitrogen). The membrane was blocked with 5% (wt/vol) milk powder at room temperature for 1 h and incubated overnight at 4°C with the primary antibody. As primary antibodies, mouse anti-Cas9 antibody (MA1-201; Invitrogen), rabbit anti-stomatin (ab166623; Abcam), and rabbit anti-vimentin as a loading control (5741; Cell Signaling Technology) were used with a dilution of 1:500 (Cas9) or 1:2,000 (stomatin and vimentin). After incubation with horseradish peroxidase (HRP)-conjugated IgG antibodies, goat anti-rabbit IgG-HRP (ab6721; Abcam) and anti-mouse IgG-HRP (7076; Cell Signaling Technology), both diluted 1:2,000, were added for 1 h at room temperature. Detection was performed using the 1-Step Ultra TMB-Blotting solution (Thermo Scientific).
9
FRTL-5 Cell Protein Extraction
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FRTL-5 cells were lysed in buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.1% NP40 and 20% glycerol at 4°C for 1 hour, followed by addition of a protease inhibitor cocktail (Complete Mini, Roche Diagnostics, Basel, Switzerland). The mixture was centrifuged at 4°C for 20 minutes to recover total cellular proteins. Protein extracts were mixed with 4× lithium dodecyl sulfate sample buffer and 10× reducing agent (Invitrogen) and incubated at 70°C for 10 minutes prior to electrophoresis. The proteins were separated on a NuPAGE 4-12% Bis Tris gel by electrophoresis and transferred to iBlot2 PVDF Mini Stacks (Invitrogen). The membranes were washed with PBS supplemented with 0.1% Tween 20 (PBST) and blocked with PBST containing 5% nonfat milk for 1 hour. Membranes were then incubated with rabbit polyclonal anti-Gapdh (1:5,000, Cell Signaling Technology) or rabbit polyclonal anti-Iyd (Dehal1) primary antibody (1:500, Abcam, Cambridge, UK) at 4°C for 12 hours, followed by a donkey anti-rabbit IgG, HRP-linked secondary antibody (1:1,000, Cell Signaling Technology) at room temperature for 1 hour. HRP was visualized using Immunostar LD reagent (Wako Pure Chemical, Osaka, Japan), and chemiluminescence was detected using the C-Digit blot scanner (LI-COR, Lincoln, NE, USA).
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