Total protein was extracted from 100 mg of agroinfiltrated N. benthamiana leaf tissue. Samples were frozen and pulverized in liquid nitrogen before homogenization in 25 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM ethylenediaminotetraacetic acid (EDTA), 0.15% IGEPAL® CA-630, 10% glycerol, 10 mM dithiothreitol, and protease inhibitor cocktail (Roche, West Sussex, UK). Crude extract was pelleted by centrifugation at 12,000× g for 4 min at 4 °C, and the clarified supernatant was collected. Coimmunoprecipitation of GFP- or RFP-tagged proteins from total leaf protein extract by incubation with GFP- or RFP-Trap magnetic agarose beads (ChromoTek, Planegg-Martinsried, Germany) was performed as previously reported [19 (link)]. Proteins were resolved on 10% polyacrylamide denaturing gels [32 (link)] and electrophoretically transferred onto a nitrocellulose membrane [33 (link)]. For immunological detection, membranes were probed with primary antibodies anti-GFP (1:1000) or anti-RFP (1:2000) (Chromotek) for 1 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Antibody binding was detected on X-ray film with Pierce ECL Substrate (Thermo-Fisher Scientific) or signals were directly captured in a G:BOX Chemi XRQ machine (Syngene, Cambridge, UK).
Igepal ca 630
Manufactured by Roche
Sourced in United Kingdom
IGEPAL® CA-630 is a non-ionic detergent primarily used as a solubilizing agent and emulsifier in various laboratory applications. It is a polyoxyethylene octylphenyl ether that can be used to facilitate the extraction and purification of proteins, enzymes, and other biomolecules from complex mixtures.
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Lab products found in correlation
Protease inhibitor, by Roche (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti gfp, by Proteintech (1 mentions)
Anti rfp, by Proteintech (1 mentions)
Pierce ecl substrate, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
A2095, by Merck Group (1 mentions)
Benzonase nuclease, by Roche (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Precellys homogenizer, by Bertin Technologies (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Gfp trap a beads, by Proteintech (1 mentions)
Mouse anti rfp, by Proteintech (1 mentions)
Rabbit anti gfp, by Proteintech (1 mentions)
9 protocols using igepal ca 630
1
Affinity Purification of GFP/RFP Proteins
2
Precipitation of Endogenous HIF-1α
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For precipitation of endogenous HIF-1α, Cezanne-depleted and control cells were treated with 20 μM MG132 for 2 h (6-well plate) and subsequently lysed in 100 μl lysis buffer per well (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% (v/v) IGEPAL® CA-630, 2 mM EDTA, 1 mM dithiothreitol, 100 mM NaF and 1 tablet/10 ml cOmplete, Mini, EDTA-free protease inhibitors (Roche)). Cleared cell lysate was rotated at 4°C for 3 h with 2 μg of anti-HIF-1α antibody (sc-10790, Santa Cruz) and Protein A Sepharose (GE Healthcare), which was added after 2 h. Immobilised antigene–antibody complex was then washed three times with PBS and eluted in 2× LDS sample buffer (Invitrogen).
3
Quantification of CSAD Protein Levels
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Western blot analysis was performed according to the method previously described [10 ]. Briefly, frozen liver and brain were homogenized with PBS buffer containing IGEPAL CA-630 and protease inhibitors (Roche Applied Science). Supernatants from tissue homogenates were separated using SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with rabbit anti-CSAD antibody and then with alkaline phosphatase labeled goat anti-rabbit secondary antibody after washing the membrane with PBS containing 0.2% Tween 20. The membrane was visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. Polyclonal rabbit anti-CSAD was produced in our laboratory using c-terminal 15 peptides obtained from Synpep Corp. (Doblin, CA) [14 (link)].
4
E2F1 Deubiquitination by Cezanne
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HEK293 cells (3×106) were seeded in 10-cm culture dishes and transfected 24 h later with 5 µg pCMV-HA-E2F1. Subsequently, cells were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) IGEPAL® CA-630, 0.2% (v/v) SDS, 10 mM NaF, 1 mM PMSF, 5 mM N-ethylmaleimide, 1× Complete EDTA-free protease inhibitors (Roche) and 1 µl/ml Benzonase® Nuclease (≥250 units/µl). Cleared lysates were incubated with 10 µl of agarose conjugated to an antibody against hemagglutinin (HA) (Sigma-Aldrich #A2095) per sample for 3 h at 4°C. Beads were washed three times with wash buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl], once with DUB buffer [50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 5 mM DTT] and equally split in two reaction tubes. Activation of 2 µg GST-Cezanne1-449 was initiated by incubating in DUB dilution buffer [150 mM NaCl, 25 mM Tris (pH 7.4), 10 mM DTT] for 10 min at room temperature. GST–Cezanne or DUB buffer alone (control) was added to immobilised HA–E2F1 and incubated for 1 h at 37°C. Subsequent SDS-PAGE and immunoblots were performed using standard protocols.
5
Detecting Ubiquitin-Linked Proteins in Cell Lysates
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Protein extracts were prepared using RIPA buffer (Sigma: 150 mmol/L NaCl, 1.0% IGEPAL® CA‐630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mmol/L Tris, pH 8.0.) and completed with protease inhibitor (Roche, Welwyn Garden City, UK). N‐ethylmaleimide (NEM; 25 mmol/L, Sigma) was also added to the RIPA buffer for detecting ubiquitin linked to immunoprecipitation of p62. Immunoprecipitation was conducted with anti‐p62 in 300‐600 μg of whole‐cell lysate overnight at 4°C. Immunoprecipitates were captured with Protein A magnetic beads. After extensive washing, bound proteins were released by boiling in SDS‐PAGE sample buffer. Protein extracts (40 µg of immunoprecipitates) were analyzed by SDS‐PAGE (Invitrogen) and detected with Western Blot analysis by chemiluminescence (ECL Plus; GE Healthcare). Protein expression levels were expressed relative to β‐actin.
6
Quantitative Proteomic Analysis of RHDV2-Infected Rabbit Liver
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RHDV2-infected and uninfected control rabbit liver samples [described in a previous study (Neave et al., 2018 (link))] were used for label-free quantification (LFQ). Briefly, rabbits were either infected with RHDV2 (genotype GI.1bP-GI.2) or mock-infected with PBS. The animals were humanely killed 24 h post-infection, and 25% liver homogenates were prepared in RNA-later buffer and stored at −20°C. Samples from three RHDV2-infected animals (K375, K376, and K378) and three uninfected animals (K3, K14, and K12) were used in this study. Liver samples were clarified at 0.2 g for 5 min at 4°C, and 100 mg of solid precipitate was collected into tubes with glass beads. Liver homogenates were lysed in 300 μl of PBS (pH 7.5) with 150 mM NaCl, 0.5 mM EDTA, 0.5% Igepal CA-630 with protease inhibitors (Roche, Basel, Switzerland), 1 mM MgCl2, and 1 μl/ml benzonase nuclease (5 KU; Sigma-Aldrich). The suspension was homogenized using a Precellys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and incubated on ice for 30 min with gentle rocking to allow for full lysis. Finally, the lysates were cleared by centrifugation and supernatants were used for MS sample preparation.
7
Nuclear Factor-κB Binding Assay
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Whole cell extracts were obtained from transfected BAEC by lysis in buffer containing 50 mM HEPES.NaOH pH7, 250 mM NaCl, 5 mM EDTA, 0.1% (v/v) Igepal CA-630, protease inhibitor tablets (Roche Applied Science). Five micrograms of cleared lysates were incubated for 30 min at room temperature with 60 000 cpm of double-stranded purified γ-32P ATP (Perkin Elmer)-labeled Ig κ light chain enhancer oligonucleotide (5′-AGT TGA GGG ACT TTC CCA GGC-3′). For competition, either non-labeled sense oligonucleotide (co Ig κB) or non-labeled scrambled oligonucleotide (5′-ACA GTA TCA AAG GCT CAC ATG-3′) (co sc Ig κB) was added. For the super-shift, complexes were incubated for 1 h on ice with 1 μg of mouse monoclonal p65 antibody (clone F-6; Santa Cruz Biotechnology). Protein–DNA complexes were separated on 5% Tris/glycine/EDTA poly-acrylamide gels, bands were visualized by autoradiography and band intensities were quantified.
8
Msd1-GFP Augmin Immunoprecipitation
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Flies expressing full-length Msd1-GFP via UASp/maternal-α-Tubulin GAL4 control (Wainman et al., 2009 (link)) were maintained according to standard procedures at 25°C. Batches of 0- to 3-h-old embryos laid by cages of 1- to10-day-old flies were dechorionated, weighed, flash frozen in N2 (l) and stored at −80°C. A total of 8 g of frozen embryos were homogenized in 16 ml C buffer (50 mM HEPES, pH 7.4, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.1% IGEPAL CA-630, Roche protease inhibitors). Extract was clarified through centrifugation at 10,000 g for 10 min, 100,000 g for 30 min, and 100,000 g for a further 10 min. Clarified extract was incubated with 50 μl GFP-TRAP-A beads (Chromotek, Germany) overnight at 4°C to immunoprecipitate Augmin. Msd1-GFP/Augmin-GFP-TRAP-A beads were washed three times with ice-cold C buffer and three times with ice-cold C buffer without IGEPAL CA-630. Based on previous semi-quantitative western blotting (not shown) we estimate 80 μg of Augmin was present in the sample.
9
Leaf Protein Extraction and Detection
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To extract total leaf protein, 100 mg of agroinfiltrated leaf tissue was ground to a powder with a pestle and liquid nitrogen. The powdered sample was homogenized in protein extraction buffer [10% glycerol, 25 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM ethylenediaminotetraacetic acid (EDTA), 0.15% IGEPAL ® CA-630 (Octylphenoxy poly(ethyleneoxy)ethanol, branched), 10 mM dithiothreitol, and protease inhibitor cocktail (Roche)] and centrifuged for 4 min at 12,000 g at 4°C, to pellet the crude extract and the supernatant collected.
Protein extracts were analyzed on an SDS-PAGE gel (46) (link) and electrophoretically transferred onto a nitrocellulose membrane (47) (link). For immunological detection of GFP-tagged and RFPtagged proteins, rabbit anti-GFP (1:1000) and mouse anti-RFP (1:2000) (ChromoTek) monoclonal primary antibodies were used in combination with anti-rabbit or anti-mouse IgG horseradish peroxidase conjugated secondary antibodies. The blotted membrane was incubated with Pierce Enhanced Chemiluminescence ECL-Plus substrate and chemiluminescence signals imaged using X-ray film (Fujifirm, Toyo, Japan).
Protein extracts were analyzed on an SDS-PAGE gel (46) (link) and electrophoretically transferred onto a nitrocellulose membrane (47) (link). For immunological detection of GFP-tagged and RFPtagged proteins, rabbit anti-GFP (1:1000) and mouse anti-RFP (1:2000) (ChromoTek) monoclonal primary antibodies were used in combination with anti-rabbit or anti-mouse IgG horseradish peroxidase conjugated secondary antibodies. The blotted membrane was incubated with Pierce Enhanced Chemiluminescence ECL-Plus substrate and chemiluminescence signals imaged using X-ray film (Fujifirm, Toyo, Japan).
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