Cell pellets were lysed in lysis buffer (150 mM NaCl, 50 mM Tris, 1% Triton-X100, 1 mM orthovanadate). Protein concentrations were calculated using Bradford assay (Bio-Rad Cat#5000006). Samples were diluted to 1× Laemmli buffer (Bio-Rad, Cat# 1610747) with 10 mM DTT. Samples were heated for 10 min at 70°C and then loaded on a 12% Mini-protean TGX gel (Bio-Rad Cat# 4561044). The gels were run for approximately 1 h at 100 V. Proteins were transferred onto a PVDF membrane (Bio-Rad, Cat# 1704156) and blocked in 5% skimmed milk in PBS-T for 1 h at room temperature. Afterwards, the membrane was incubated at 4°C overnight with primary antibodies (1:1000) in 1% milk in PBS-T. We used primary antibodies as above. After washing and addition of secondary HRP-conjugated antibodies, membranes were developed using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific Cat# 32132). Membranes were stripped (Restore plus Western blot stripping buffer Thermo Scientific Cat# 46430) and re-probed using a second set of control primary antibodies. Blots were run and visualized using a Chemidoc Touch V3 Western workflow (Bio-Rad, Cat# 1708381).
Restore plus western blot stripping buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Macao, United Kingdom
Restore PLUS Western Blot Stripping Buffer is a laboratory reagent designed to remove primary and secondary antibodies from western blot membranes, allowing for the reuse of the same membrane for subsequent antibody detection.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Ripa buffer, by Merck Group (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Clarity western ecl substrate, by Bio-Rad (9 mentions)
Protease inhibitor cocktail, by Roche (8 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (7 mentions)
Image lab software, by Bio-Rad (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (6 mentions)
Ecl plus, by Thermo Fisher Scientific (6 mentions)
Chemidoc mp imaging system, by Bio-Rad (5 mentions)
Trans blot turbo transfer system, by Bio-Rad (5 mentions)
Mini protean tgx gel, by Bio-Rad (5 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (4 mentions)
Laemmli buffer, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
161 protocols using restore plus western blot stripping buffer
1
Western Blot Protein Detection Workflow
2
Western Blot for RBM20 Protein
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Protein lysates were obtained with RIPA buffer containing protease inhibitors. Following clarification of the lysate by centrifugation and assessment of protein centrifugation by BCA assay, samples were diluted in Laemmli buffer and boiled. Twenty micrograms of protein were electrophoretically separated using 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). Proteins were transferred to PVDF membranes and blocked in PBS with 0.1% Tween-20 (PBST) and 5% bovine serum albumin (BSA). The anti-RBM20 rabbit polyclonal primary antibody (ThermoFisher #PA5-57404) was diluted at 1:500 in PBST 1% BSA and incubated overnight at 4 °C. Membranes were washed three times in PBST for 10 min at room temperature, incubated for 1 h at room temperature with goat anti-rabbit HRP-conjugated secondary antibody, and washed three times in PBST for 10 min. Chemiluminescent reaction was initiated by incubation with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher), and images were acquired using a ChemiDoc Imaging System (Bio-Rad) in “high resolution” mode. Before re-probing for the housekeeping protein GAPDH (mouse monoclonal [6C5] diluted at 1:5000; Abcam #8245) according to the same protocol but using goat anti-mouse HRP-conjugated secondary antibody, membranes were treated with Restore Plus western blot stripping buffer (ThermoFisher), washed three times, and re-blocked.
3
Western Blot Standardized Protocol
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LPMs were sorted as for microarrays, pooled from multiple mice and lysed at a concentration of 25 million cells/mL using the RIPA lysis buffer system (Santa Cruz Biotechnology). Laemmli buffer (Bio-Rad, with added 2-mercaptoethanol) was added to the samples, which were then boiled for 10 minutes, and run on a Bio-Rad Miniprotean TGX gel with Precision Plus Dual Color molecular weight standards (Bio-Rad). Proteins were transferred to a BioBlot polyvinylidine fluoride membrane (Costar). Blots were blocked for 1–2 hours with 5% milk, followed by overnight staining with primary antibodies in 5% milk at 4 °C with shaking. After washing, blots were stained with horseradish peroxidase-conjugated secondary antibodies for 45–60 minutes at RT with shaking. After washing, blots were developed with either Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher) or Clarity Western ECL Substrate (Bio-Rad). Images were captured on a Chemidoc system (Bio-Rad) and inverted on Adobe Illustrator for presentation. Blots were stripped with Restore PLUS Western Blot Stripping Buffer (ThermoFisher). Blots were then washed, reblocked and restained. Antibodies used are listed in Supplementary Table 1 .
4
Western Blot Analysis of Protein Markers
5
Western Blot Protocol for Protein Analysis
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Protein concentrations in cell and tumor lysates were quantified using the DC Protein Assay (Bio-Rad,). 20 μg of proteins were mixed with Laemmli buffer, boiled for 5 min, resolved by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). Membranes were blocked with 3% BSA for 1 hour and incubated with primary antibodies (4C, 16 hours). After 3 TBS/T washes, membranes were incubated with HRP-labeled secondary antibodies (Promega) for 1 hour at room temperature. Protein bands were developed using ECL Plus Detection Reagents (GE Healthcare). To analyze multiple proteins on the same membrane, membranes were washed with Restore PLUS Western Blot Stripping Buffer (Thermo Scientific) according to the manufacturer's protocol.
6
Western Blot Analysis of Crry and EGFP
7
Protein Lysate Preparation from Zebrafish Tumors
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Protein lysates were prepared with mCherry expressed tumors from nf1a+/−; nf1b−/−; p53m/m fish. Briefly, mCherry-positive tumor mass were harvested from tricaine-anesthetized tumor fish, and were lysed on ice in 1 × RIPA (Cell Signaling, Danvers, MA, USA) containing, PhosSTOP phosphatase inhibitor cocktail tablet (Roche, Indianapolis, IN, USA) and complete protease inhibitor tablet (Roche). The inhibitors were prepared following the manufacturer's recommendation. Protein lysates were separated by gel electrophoresis, transferred to PVDF membranes and probed overnight at 4 °C with the following primary antibodies: anti-PDGFRA (Cell Signaling; 5241; 1:500), anti-p-tyrosine (EMD Millipore, Billerica, MA, USA; 05-321; 1:1000), anti-β-tubulin (Cell Signaling 2146; 1:2000), anti-AKT (Cell Signaling; 9272; 1:1000), anti-p-AKT (Cell Signaling; 4060; 1:1000), anti-p-ERK1/2 (Cell Signaling; 4377;1:1000) and anti-ERK1/2 (Cell Signaling; 9102; 1:1000). Primary antibody binding was visualized on X-ray film using anti-mouse-HRP (Cell Signaling; 7076; 1:10 000) or anti-rabbit-HRP (Cell Signaling; 7074; 1:10 000) secondary antibodies along with SuperSignal West Dura or Femto (Thermo Fisher Scientific, Waltham, MA, USA) chemiluminescent substrates. Stripping was performed using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) according to the manufacturer's protocol.
8
Western Blot Analysis of Protein Samples
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Protein samples were mixed with an equal amount of 2× Laemmli reducing buffer and boiled for 10 min before being subjected to SDS-PAGE analysis on 12% polyacrylamide gels. Proteins were stained with InstantBlue solution (Expedeon) for visualization or were transferred onto PVDF membranes using iBlot Dry Blotting System (Thermo Fisher Scientific) at 20 V for 7 min. Following protein transfer, the PVDF membranes were immediately blocked with 1× casein blocking buffer (Sigma-Aldrich) and were probed with either horseradish peroxidase (HRP)-conjugated primary antibodies against His tag (Qiagen) or with rabbit primary antibodies against HA-tag (Sigma-Aldrich) followed by anti-rabbit HRP-conjugated secondary antibodies. Membranes were thoroughly rinsed with PBS containing 0.1% Tween 20 between the incubations. Chemiluminescent signals were detected using Amersham ImageQuant 800 System (GE Healthcare) after a brief incubation of the PVDF membranes with Immobilon Forte Western Chemiluminescent HRP Substrates (EMD Millipore). For reblotting with a different antibody after image acquisition, the membranes were incubated with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) for 30 min at room temperature, rinsed thoroughly before being blocked with casein blocking buffer, and probed with antibodies as described above.
9
Western Blot Analysis of Signaling Proteins
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Western blots were performed following the method from another literature published by Novartis58 (link): total cell lysates were prepared by direct lysis of cells with RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (Thermo Fisher Scientific) with incubation on ice for 20 min and followed by centrifugation at 12,000×g at 4 °C for 5 min. The supernatant was collected and protein concentration was determined using Pierce BCA protein assay kit (#23225, Thermo Fisher Scientific). Equal amounts of proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and probed by the following antibodies with indicated dilutions: phospho-ERK1/2 (T202/Y204) (9101, Cell Signaling Technology, 1:1000), total ERK1/2 (9102, Cell Signaling Technology, 1:1000), SHP2 (3397, Cell Signaling Technology, 1:1000), phospho-STAT1 (Y701) (9167, Cell Signaling Technology, 1:1000), total STAT1 (14994, Cell Signaling Technology, 1:1000), Vinculin (13901, Cell Signaling Technology, 1:1000). After detection of phospho-STAT1 and phospho-ERK1/2, the PVDF membranes were incubated with Restore PLUS Western Blot Stripping Buffer (#46430, Thermo Fisher Scientific) before probing for levels of total STAT1 and total ERK1/2.
10
Purification and Western Blotting of CD4+ T Cells
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Leukocytes were isolated from SGs and spleens as described previously (Stacey et al., 2011 (link)). CD4+ T cells were purified via magnetic separation using a MagniSort Mouse CD4 Positive Selection Kit (Thermo Fisher Scientific). Cell lysates were generated from equal numbers of cells using NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) supplemented with 100 mM dithiothreitol (Sigma-Aldrich). Samples were loaded onto 4–12% NuPAGE Bis-Tris Gels (Thermo Fisher Scientific) after boiling and transferred to a PVDF membrane using an XCell II Blot Module (Thermo Fisher Scientific). Blots were probed with anti-arginase-1 (rabbit polyclonal, Thermo Fisher Scientific) and developed using anti-rabbit IgG–HRP (Bio-Rad). Band intensity was determined using a G:BOX Gel Imaging System (Syngene). Blots were then stripped using Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) and probed again with anti-actin (rabbit polyclonal, Abcam).
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