To probe for expression of the HA-tagged MetRSNLL, HFFs infected with either WT T. gondii or the Tg-MetRSNLL strain were lysed in 1× sodium dodecyl sulfate (SDS) sample buffer and resolved on 8% SDS-polyacrylamide gels. The samples were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). After the transfer, the membranes were blocked in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST) and 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) or 5% nonfat dry milk for 1 h. After being blocked, the membrane was incubated in anti-HA-peroxidase (Roche, Mannheim, Germany) antibody at a 1:4,000 dilution for 30 min. After incubation, the blot was washed 3 times with TBST and developed using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL). To test for equal loading, the blot was stripped with Restore Western blot stripping buffer (Thermo Scientific, Rockford, IL) and reprobed with mouse anti-Tg-SAG1 (GenWay) at a 1:4,000 dilution for 1 h. After three washes with TBST, the blot was incubated with goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:4,000 dilution for 1 h. After the blot was washed 3 times with TBST, it was developed by using the SuperSignal West Pico chemiluminescent substrate.
Anti ha peroxidase
Manufactured by Roche
Sourced in United States
Anti-HA-peroxidase is a laboratory reagent used for the detection and quantification of proteins tagged with the haemagglutinin (HA) epitope. It is a conjugate of an anti-HA antibody and the enzyme peroxidase, which catalyzes a colorimetric reaction for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (3 mentions)
Anti pgk1, by Thermo Fisher Scientific (3 mentions)
Immobilon p, by Merck Group (3 mentions)
Anti ha affinity matrix, by Roche (2 mentions)
Pser36 shc1, by Abcam (2 mentions)
Anti flag m2 antibody, by Merck Group (2 mentions)
Las4000 system, by GE Healthcare (2 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Santa Cruz Biotechnology (1 mentions)
Anti xbp1, by Cell Signaling Technology (1 mentions)
Irdye 800cw conjugated secondary antibody, by LI COR (1 mentions)
Mg132, by Fujifilm (1 mentions)
Anti eif5a, by BD (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Amersham imager, by GE Healthcare (1 mentions)
32 protocols using anti ha peroxidase
1
Western Blot Analysis of HA-tagged MetRS
2
Whole-Cell Lysate Preparation and Immunoblotting
3
Co-immunoprecipitation of MYB12-HA and NtbHLH1/NtWD40-1 in Narcissus
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The combination of constructs expressing the corresponding MYB12-HA-tagged proteins and NtbHLH1-CFP or NtWD40-1-GFP-tagged proteins were co-transformed into narcissus protoplasts. After incubation 16 h, the transfected protoplasts were harvested for the Co-IP and immunoblotting assays as described previously (Zheng et al. 2018 (link)). The HA-tagged proteins were immunoprecipitated with anti-HA antibody coupled beads (anti-HA affinity matrix, Roche). The immunoprecipitated proteins were then separated via 13.5% SDS-PAGE, blotted and analyzed by immunoblotting with anti-GFP (TransGen, HT801, 1:1,000 dilution) and Anti-HA-Peroxidase (Roche, 3F10, 1:500 dilution) antibodies.
4
Proteasome Inhibition and Western Blotting
5
Western Blot Protein Detection
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Proteins from whole-cell extracts were resolved on 4%–20% SDS-PAGE (4561096; Bio-Rad) followed by transfer of the proteins to 0.2-μm nitrocellulose membranes (Bio-Rad). The membranes were blocked in 5% nonfat milk in TBS, Tween 20 for 1 h and immunoblotted with antibodies against GFP (mouse anti-GFP.1, 4°C overnight, 632381 [Takara Bio, Clontech] or rabbit anti-GFP.2, 1 h at room temperature [gift from M.P. Rout; Cristea et al., 2005 (link)]), or against HA (anti-HA-peroxidase, 1 h at room temperature, 12013819001; Roche). Blots were incubated with HRP-conjugated secondary antibodies (1 h at room temperature; goat anti-mouse IgG, 31430, or goat anti-rabbit IgG, 31460; Invitrogen) and visualized by ECL (Thermo Fisher Scientific) using a VersaDoc Imaging System (Bio-Rad). Relative protein loading was visualized using Ponceau S Solution (Sigma-Aldrich).
6
Protein Expression in Arabidopsis
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Sl06g050500, Sl03g007310, and Sl08g076960 coding sequences in the pCR8/GW/TOPO entry clone were recombined by LR reaction into the Gateway-compatible ALLIGATOR2 vector (Bensmihen et al., 2004 (link)). The ALLIGATOR2 vector drives expression of the recombined gene under control of the Cauliflower mosaic virus (CaMV) 35S promoter and introduces a triple haemagglutinin (HA) epitope at the N-terminus of the encoded protein. Selection of transgenic lines is based on the visualization of green fluorescent protein (GFP) in seeds, whose expression is driven by the specific seed promoter At2S3. The ALLIGATOR2 constructs were transferred to Agrobacterium tumefaciens C58C1 (pGV2260) (Deblaere et al., 1985 (link)) by electroporation and used to transform Columbia wild type by the floral dip method. T1 transgenic seeds were selected based on GFP visualization and sown in soil to obtain the T2 generation. Homozygous T3 progeny was used for further studies, and expression of HA-tagged protein was verified by immunoblot analysis using anti-HA-peroxidase (Roche).
7
Flag-tagged Protein Pulldown Assay
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Lysates from HeLa cells cotransfected with plasmids pIZ2047 and pIZ3423, expressing SseK1-3xFlag and 3xHA-TBCB, respectively, were incubated for two hours with monoclonal anti-FLAG antibodies, overnight with protein A/G plus-agarose beads (Santa Cruz Biotechnology, Dallas, TX, USA), and then centrifuged. The beads were washed five times in NP40 buffer. Proteins were eluted and dissolved into Laemmli sample buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 0.0005% bromophenol blue) containing 5% β-mercaptoethanol, incubated at 95 °C for 5 min and subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose filter and probed with anti-FLAG (Sigma) and anti-HA-peroxidase (clone 3F10, Roche).
8
Protein Extraction and Immunoblot Analysis
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Freshly harvested and grinded 5-day-old seedlings or 5 × 105 transfected protoplasts were lysed with 50 µL of extraction buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1% (w/v) Triton X-100, 5 mM DTT, PhosSTOP phosphatase inhibitor cocktail (Roche), 1x EDTA free-Complete Protease Inhibitor Cocktail (Roche). After vortexing vigorously for 30 s, the samples were centrifuged at 12,000 × g for 10 min at 4 °C and supernatants were collected. For immunoblot analysis, 25 µL of collected supernatants were separated by SDS-PAGE and subjected to immunoblotting using anti-GFP-HRP antibody (130-091-833, Miltenyi Biotec, dilution 1:5000) and Anti-HA-Peroxidase, (3F10, Roche, dilution 1:5000).
9
Quantifying Cellular Ubiquitin Conjugates
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To analyze total ubiquitin conjugates, whole-cell extracts were prepared from exponential-phase cultures. Cells were normalized by optical density; cell pellets were resuspended in 1× Laemmli loading buffer (LLB) and boiled for 5 min. For all other immunoblots, extracts were prepared by a lithium acetate/sodium hydroxide method as follows. Exponential-phase cultures were normalized by optical density, treated with 2 M lithium acetate on ice for 5 min, and then treated with 0.4 M sodium hydroxide on ice for 5 min. Cell pellets were resuspended in 1× LLB and boiled for 5 min. Analysis was by standard SDS–PAGE followed by immunoblotting. For cycloheximide chase analyses, cycloheximide (100 µg/ml) was added at time 0 to inhibit protein synthesis, and extracts were prepared as described at the indicated time points. Note that the experiments of Figure 3, A–C and E , were carried out at 37°C. Where indicated, the unfolded protein response was induced by treating cells with tunicamycin at 5 μg/ml for 1 h.
The following antibodies were used: anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Pgk1 (459250; Invitrogen), anti–HA-peroxidase (12013819001; Roche), anti-actin (MA511866; ThermoFisher), anti–phospho-eIF2α (9721S; Cell Signaling Technology), and anti-Kar2 (sc-33630; Santa Cruz Biotechnology).
The following antibodies were used: anti-ubiquitin (SC-8017; Santa Cruz Biotechnology), anti-Pgk1 (459250; Invitrogen), anti–HA-peroxidase (12013819001; Roche), anti-actin (MA511866; ThermoFisher), anti–phospho-eIF2α (9721S; Cell Signaling Technology), and anti-Kar2 (sc-33630; Santa Cruz Biotechnology).
10
Protein Extraction and Western Blotting Workflow
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Cells were harvested, and proteins were extracted using cell lysis buffer (Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor cocktail. The protein concentration was measured with a BCA kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The primary antibodies and their concentrations were as follows: anti-vinculin (Abcam, 1:1000), anti-RORα (Abcam, ab60134, 1:1000), anti-RORγ (Proteintech, 13205-1-AP, 1:1000), anti-c-myc (Abcam, ab32072, 1:1000), anti-HA-Peroxidase (Roche, 12013819001, 1:1000) and anti-NEDD4 (Cell Signaling Technology, CST, #2740, 1:1000). Western blotting was performed as described previously [55 (link)]. The gray values of the immunoblots were measured by ImageJ (version 1.48) software (Wayne Rasband, National Institutes of Health, USA). The statistical differences for all the western bot data were shown in Supplementary Figs. 6 , 7 .
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