For Western blot analysis, protein lysates from yeast meiotic cultures were prepared using TCA precipitation and run on 8% or 10% SDS gels, transferred for 90 min at 300 mA and blotted with the selected antibodies, as described (Kuhl et al., 2020 (link)). Primary antibodies with respective dilutions were used: rabbit α-Hop1 (made in-house; 1:10,000), rabbit α-Mer240-271 (made in-house; 1:10,000); mouse α-Pgk1 (Thermo Fisher, 1:5000); rabbit α-phospho-Histone-H3-Thr11 (Abcam, 1:1000), mouse α-HA (Biolegend, diluted 1:500), α-Myc-9E11 (Abcam, ab56, 1:1000).
Mouse α ha
Manufactured by BioLegend
Sourced in United States, Germany
Mouse α-HA is a primary antibody that specifically binds to the influenza hemagglutinin (HA) epitope tag. It is a useful tool for the detection and purification of HA-tagged recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors, by Boehringer Ingelheim (1 mentions)
Mouse α tubulin, by Merck Group (1 mentions)
Rabbit α pttg1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Alliance 2, by Uvitec (1 mentions)
Digitonin, by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Goat α mouse hrp, by Jackson ImmunoResearch (1 mentions)
Goat α rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Protein a agarose, by Repligen (1 mentions)
Poly l lysine (pll), by Peptide Institute (1 mentions)
Goat anti rat hrp, by Cell Signaling Technology (1 mentions)
Goat α rat igg cy2, by Jackson ImmunoResearch (1 mentions)
Goat α mouse igg alexa657, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Mouse α pdi, by Thermo Fisher Scientific (1 mentions)
Mouse α dnak, by Stressgen (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
5 protocols using mouse α ha
1
Yeast Meiotic Protein Analysis
2
Protein Expression Analysis Protocol
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For Western blot, cells were lyzed in RIPA buffer (50 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA). Proteins were resolved by SDS-PAGE and then transferred onto PVDF membranes (Millipore). All buffers contained a cocktail of protease inhibitors (Boehringer, Ingelheim am Rhein, Germany). Membranes were developed using the enhanced chemiluminescence (ECL westar, Cynagen, Bologna, Italy). Bands were analyzed by chemiluminescence imaging system, Alliance 2.7 (UVITEC, Cambridge, UK) and quantified by the software Alliance V_1607.
The following primary antibodies were used: rabbit α-PTTG1 (Abcam), mouse α-MMP-2 (Thermo Fisher, Waltham, MA, USA), mouse α-HA (BioLegend, San Diego, CA, USA), mouse α-tubulin (Sigma, St. Louis, MO, USA), mouse α-actin monoclonal antibody C-40 (Sigma), and rabbit α-Sp1 (Santa Cruz, Santa Cruz, CA, USA).
The following primary antibodies were used: rabbit α-PTTG1 (Abcam), mouse α-MMP-2 (Thermo Fisher, Waltham, MA, USA), mouse α-HA (BioLegend, San Diego, CA, USA), mouse α-tubulin (Sigma, St. Louis, MO, USA), mouse α-actin monoclonal antibody C-40 (Sigma), and rabbit α-Sp1 (Santa Cruz, Santa Cruz, CA, USA).
3
Immunoblotting Antibodies and Reagents
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Antibodies used for immunoblotting are as follows: Rat α-FLAG L5 (#637303; BioLegend), Rabbit α-HA, Rabbit α-GFP, Rabbit α-Sec62, Rabbit α-Sec63, Rabbit α-Sec61β, Rabbit α-BAG6, and Rabbit α-Sec61α (Chitwood et al., 2018 (link); Mariappan et al., 2010 (link); Snapp et al., 2004 (link)) are a gift from Dr. Ramanujan Hegde (MRC Laboratory of Molecular Biology, Cambridge, UK). Rabbit α-BiP (#11587-1-AP; Proteintech), Mouse α-HA (#901513; Biolegend), Mouse α-PDI (#MA3-018; Affinity Bioreagents), Goat α-Rat-HRP (#7077; Cell Signaling), Goat α-Mouse-HRP (#115-035-003; Jackson ImmunoResearch), Goat α-Rabbit-HRP (#111-035-003; Jackson ImmunoResearch), Goat anti-rat HRP (#7077S; Cell signaling), Goat α-RAT IgG-Cy2 (#112-225-167; Jackson ImmunoResearch), Goat α-Mouse IgG-Alexa657 (#A-21235; Invitrogen). Beads were purchased as follows: Strep-Tactin XT beads (#2-4010-010; IBA), Rat anti-FLAG L5 affinity gel (#651503; Biolegend), Protein A agarose (#CA-PRI-0100; Repligen), Mouse anti-HA magnetic beads (#88837; Pierce), Poly L-lysine (#OKK-3056; Peptides International). Rabbit Reticulocyte Lysate was purchased from Green Hectares (Ph:1-800-GHLYSAT). Detergents were purchased as follows: digitonin (EMD Millipore), Triton X-100 (Thermo Fisher Scientific), sodium deoxycholate (Sigma-Aldrich), SDS (Sigma-Aldrich), and Tween 20 (American Bioanalytical).
4
Immunoblot Analysis of S. sonnei
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S. sonnei cultures were induced with 1 mM IPTG at OD600=0.2. After a further 60 min of growth, liquid cultures were harvested and concentrated to an OD600=5.0. The cell pellet (cellular fraction) was resuspended in reducing Laemmli buffer and heated at 95 °C for 5 min. The supernatant fraction was filter sterilised with a 0.22 µm filter and precipitated with trichloroacetic acid. The precipitated supernatant faction was resuspended in reducing Laemmli buffer and heated at 95 °C for 5 min. All samples were separated on a 15 % SDS-PAGE and proteins transferred to PVDF membranes. Membranes were probed with mouse α-HA (Biolegend) or mouse α-DnaK (Stressgen). Followed by an anti-mouse HRP conjugated secondary antibody (Sigma Aldrich). Membranes were then incubated with ECL reagent and visualised on a BioRad ChemiDoc XRS+system.
5
Co-immunoprecipitation of LeEIX2-HA and SlDRP1B-GFP
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Co-immunoprecipitation assays were performed as described by Leibman-Markus [47 (link)]. N. benthamiana leaves transiently co-expressing LeEIX2-HA and SlDRP1B-GFP were harvested 40 h after infiltration. Leaf petioles were immersed in EIX 3 μg/mL (or water as mock) for seven minutes and then transferred to the water for an additional seven minutes. A total of 500 mg leaf tissue was used for co-immunoprecipitation, with 13 μL α-HA Affinity Matrix (Roche, Indianapolis, ID, USA). Samples were run in SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with antibodies as required: rat α-GFP (Chromotek, Planegg-Martinsried, Germany) and mouse α-HA (Biolegend, San Diego, CA, USA).
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