Cell fractionation analysis was conducted on entire seedlings of HA-tagged ATL2-OX4-1 transgenic plants. The tissue was ground in liquid nitrogen and suspended in an extraction buffer (50 mM Tris-MES, pH 8.0, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 10 mM EGTA, 10 mM ascorbic acid, 5 mM DTT, protease inhibitor cocktail; Roche, Basel, Switzerland) on ice, using a previously outlined method [55 (link)]. The resulting total protein extracts were centrifuged at 10,000× g for 10 min, and the supernatant was recovered and subjected to centrifugation at 125,000× g for 1 h to separate the membrane fraction from insoluble materials. The membrane fraction was reconstituted in a detergent-free buffer (5 mM potassium phosphate (pH 7.8), 2 mM DTT, with protease inhibitor cocktail; Roche). Portions of each sample were combined with a SDS-PAGE loading buffer for protein gel analysis. SDS gel electrophoresis was used to separate total proteins, and Western blots were probed with anti-HA monoclonal antibodies (Roche, Basel, Switzerland). Coomassie brilliant blue R250 (CBB) was applied to stain the gels for use as a loading control.
Anti ha monoclonal antibody
Manufactured by Roche
Sourced in Switzerland, China
The Anti-HA monoclonal antibody is a laboratory reagent used to detect and purify proteins that are tagged with the HA (Hemagglutinin) epitope. The antibody specifically binds to the HA tag, allowing for the identification and isolation of the tagged proteins from complex samples.
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Lab products found in correlation
Anti flag m2 monoclonal antibody, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti β actin monoclonal antibody, by Merck Group (2 mentions)
Anti human galnt6 polyclonal antibody, by Merck Group (2 mentions)
Monoclonal anti α tubulin antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Anti grp78, by Proteintech (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Chitin, by Merck Group (1 mentions)
Ecl prime detection reagent, by GE Healthcare (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Anti atf6, by Cell Signaling Technology (1 mentions)
Anti ire1α, by Cell Signaling Technology (1 mentions)
Anti parp1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 7, by Cell Signaling Technology (1 mentions)
15 protocols using anti ha monoclonal antibody
1
Cell Fractionation Analysis of ATL2-OX4-1
2
Western Blot Analysis of eIF2α Phosphorylation
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Western blotting was performed as previously described (Hibi et al., 2018 ). To detect eIF2α phosphorylation, phospho‐eIF2α (Ser52) polyclonal antibody (Thermo Fisher Cat#44‐728G), eIF2α, eIF2α polyclonal antibodies (Invitrogen Cat#PA5‐41916), tubulin, monoclonal anti‐α‐tubulin antibody (Sigma Cat#T6074), and Ecl1‐HA, anti‐HA monoclonal antibodies (Roche Cat#11 583 816 001) were used.
3
Immunoprecipitation of Rad1-Rad10 Complex
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RAD1 and rad1R218A were tagged with a C-terminal 3XHA tag at their genomic locus. Yeast lysates expressing Rad1–3HA and rad1R218A-3HA were prepared by lysing cells with glass beads in 0.6 ml cold IP150 solution (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche Life Science). Anti-HA monoclonal antibody (Roche Life Science) was added to pre-cleared cell lysate and incubated at 4°C for 90 min. A 50% slurry of Protein A/G Agarose (30 μl) was added to the lysate to immunoprecipitate Rad1–Rad10 or rad1R218A–Rad10, and the mixture was incubated for an additional 30 min at 4°C. The beads were collected by centrifugation and washed extensively with IP150. The bound protein was eluted by the addition of 1× Laemmli buffer and incubation at 95°C. The eluted proteins were separated by SDS-PAGE, transferred to nitrocellulose. The presence of RPA was detected by immunoblotting using α-Rpa1 polyclonal antibody.
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RAD1 and rad1R218A were tagged with a C-terminal 3XHA tag at their genomic locus. Yeast lysates expressing Rad1–3HA and rad1R218A-3HA were prepared by lysing cells with glass beads in 0.6 ml cold IP150 solution (20 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche Life Science). Anti-HA monoclonal antibody (Roche Life Science) was added to pre-cleared cell lysate and incubated at 4°C for 90 min. A 50% slurry of Protein A/G Agarose (30 μl) was added to the lysate to immunoprecipitate Rad1–Rad10 or rad1R218A–Rad10, and the mixture was incubated for an additional 30 min at 4°C. The beads were collected by centrifugation and washed extensively with IP150. The bound protein was eluted by the addition of 1× Laemmli buffer and incubation at 95°C. The eluted proteins were separated by SDS-PAGE, transferred to nitrocellulose. The presence of RPA was detected by immunoblotting using α-Rpa1 polyclonal antibody.
4
Immunocytochemical Analysis of Intracellular Proteins
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Immunocytochemistry was performed as previously described [17] (link), [18] (link). The first antibodies used in this study included: anti-Flag M2 monoclonal antibody (1:500, Sigma-Aldrich), anti-HA monoclonal antibody (1:500, Roche), anti-GRP78 (1:25, Proteintech) and anti-PDI monoclonal antibody (1:100, Cell signaling). The secondary antibodies were: Alexa Fluor 488 Anti-Mouse or -Rabbit IgG antibodies (1:500–1000, Life Technologies) and Alexa Fluor 594 Anti-Rat or -Mouse IgG antibodies (1:500–1000, Life Technologies). Finally, cells were stained with DAPI (Vector Laboratories) and examined by TCS SP5 Confocal Laser Scanning Microscope (Leica Microsystems).
5
Western Blot Protein Detection Protocol
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Western blot was performed as described previously [14] (link). Protein bands were visualized by ECL detection reagents (GE Healthcare). The primary antibodies used in this study were as follows: anti-human ER-α monoclonal antibody (1:400, Santa Cruz), anti-human GALNT6 polyclonal antibody (1:500, Sigma-Aldrich), anti-Flag M2 monoclonal antibody (1:1000, Sigma-Aldrich), anti-HA monoclonal antibody (1:1000, Roche), and anti–β-actin monoclonal antibody (1:10,000, Sigma-Aldrich). The secondary antibodies were goat anti-rabbit or anti-mouse IgG-HRP antibodies (1:10,000-1:30,000, Santa Cruz). For the detection of O-glycosylated proteins, we performed lectin blotting using biotinylated Vicia villosa agglutinin (VVA) lectin (1:1000, Vector Laboratories) and Streptavidin-HRP (1:10,000, Thermo Scientific).
6
Monitoring ATL2 Protein Dynamics
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Arabidopsis seedlings expressing the HA-tagged ATL2 or ATL2C138A protein under the regulation of the CaMV35S promoter were germinated and cultivated on selective media for 10 days (with a light/dark cycle of 16:8 h). Following this, the seedlings were transferred to half-strength MS liquid medium containing MG132 (Sigma-Aldrich, Saint Louis, MO, USA) and/or cycloheximide (Sigma-Aldrich, Saint Louis, MO, USA), and/or chitin (Sigma-Aldrich, Saint Louis, MO, USA) at different concentrations. The treated seedling samples were collected at predetermined times for subsequent protein expression analysis using immunoblotting. In immunoblotting, the seedlings underwent extraction in a denaturing buffer—50 mM Tris/HCl pH 6.8, 4% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol, and 0.001% (w/v) bromophenol blue—at a ratio of 20 volumes (w/v). Following a 3 min incubation at 100 °C, the extracts were filtered via centrifugation at 8000× g for 3 min. The total proteins were then separated via SDS gel electrophoresis, and Western blots were subjected to probing with the anti-HA monoclonal antibody (Roche, Basel, Switzerland). Coomassie brilliant blue R250 was utilized to stain the gels, and HSP90 (90 kDa) served as the designated loading control.
7
Western Blot Analysis of Protein Expression
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Western blot was performed as described previously [17] (link). Finally, protein bands were visualized by ECL or ECL prime detection reagents (GE Healthcare). The primary antibodies used in this study were: anti-human GRP78 polyclonal antibody (1:1000, Santa Cruz), anti-human GALNT6 polyclonal antibody (1:1000, Sigma-Aldrich), anti-Flag M2 monoclonal antibody (1:1000, Sigma-Aldrich), anti-HA monoclonal antibody (1:1000, Roche), anti-PARP-1 antibody (1:1000, Santa Cruz), anti-caspase 7 (1:1000, Cell signaling), anti-PERK (1:1000, Cell signaling), anti-IRE1α (1:1000, Cell signaling), anti-ATF6 (1:1000, Cell signaling), and anti-β-actin monoclonal antibody (1:10,000, Sigma-Aldrich). The secondary antibodies were goat anti-rabbit, anti-rat, and anti-mouse IgG-HRP secondary antibodies (1:10,000~ 1:30,000, Santa Cruz). Intensity of protein band was quantified by ImageJ software as previously described [8] (link).
8
Antibody Application for Western Blot and Immunofluorescence
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The following antibodies were used: anti-Myo1b polyclonal antibody (1:1,000 for Western blot; 1:50 for immunofluorescence; Almeida et al., 2011 (link)); anti-EphB2 polyclonal antibody (0.5 µg/ml; R&D Systems); anti-GFP mouse monoclonal antibody (1:1,000 for Western blot; Roche); anti-phosphotyrosine mouse monoclonal antibody (1:1,000 for Western blot; clone 4G10); anti-HA monoclonal antibody (1:400; 3f10; Roche); anti-tubulin monoclonal antibody (1:5,000; Sigma-Aldrich); anti-pMLC (ser19; 1:1,000; Cell Signaling Technology); anti-NM myosin heavy chain II (polyclonal antibody; 1:2,000; Covance); and Alexa- and horseradish peroxidase–conjugated secondary antibodies (1:500; Invitrogen; 1:5,000; Jackson ImmunoResearch Laboratories, Inc.; 1:500; Molecular Probes). Alexa-conjugated phalloidin was used to detect F-actin (1:500; Invitrogen).
9
Western Blot Analysis of Tagged Yeast Proteins
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Western blot analysis was carried out using 25 µg of total protein extracts from HA-epitope tagged yeast strains. Protein samples were separated with 12% sodium dodecyl sulphate (SDS-) PAGE and subsequent blotting on a PVDF membrane (Millipore). The membrane was blocked with 5% nonfat dry milk. For the detection of tagged proteins anti-HA monoclonal antibody (Roche; 1:1000 dilution) were used following incubation with anti-mouse-IgG-conjugated horseradish peroxidase (BioRad; 1:10000 dilution). Pyk1 was detected using polyclonal anti-Pyk1(1:10000) followed by anti-rabbit-IgG-conjugated horseradish peroxidase (BioRad: 1:10000 dilution).
10
Immunoblot Analysis of FvC5SD
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Total protein was resolved on 12.5% SDS PAGE and electro transferred to nitrocellulose membrane. For detecting endogenous FvC5SD from F. velutipes, peptide based polyclonal antibody raised in rabbit against FvC5sdp (in 1:10,000 dilution) and alkaline phosphatase conjugated anti-rabbit secondary antibody (1:20,000) were used. Anti- HA monoclonal antibody (Roche) was used at dilution of 1:1000 to detect HA tagged FvC5SD in S. pombe. Protein bands were visualized using Horse radish peroxidase-conjugated (Amersham) secondary antibodies.
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