Antibodies were purchased from Cell Signaling Technology unless indicated otherwise and are listed with details in Table S1 . Anti-HA peroxidase-conjugated antibody (final concentration, 25 mU/mL) was purchased from Sigma-Aldrich (clone 3F10). All other reagents and media were purchased from Sigma-Aldrich unless otherwise stated.
Anti ha peroxidase conjugated antibody
Manufactured by Merck Group
Sourced in United States
The Anti-HA peroxidase-conjugated antibody is a laboratory tool used for the detection and identification of proteins tagged with the HA (Hemagglutinin) epitope. The antibody is conjugated with the enzyme peroxidase, which enables the visualization of the target protein through a colorimetric or chemiluminescent reaction.
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Lab products found in correlation
5 protocols using anti ha peroxidase conjugated antibody
1
Antibody Purchase and Reagent Details
2
Yeast Two-Hybrid Assay for RsRlpA Interactions
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For the yeast-two-hybrid assay, full-length cDNA from the RsRlpA gene was ligated to the pGADT7 prey vector (Takara, Kusatsu, Japan) and plant proteases to the pGBKT7 bait plasmid (Takara, Kusatsu, Japan) and simultaneously transformed to the Saccharomyces cerevisiae AH109 strain. Transformations with empty vectors were used as negative controls. For co-immunoprecipitation assays, the RsRplA GFP-tagged protein and HA-tagged plant protease were transiently co-expressed in N. benthamiana and pull-downed as described above. GFP-tagged protein was detected using the B2 anti-GFP HRP-conjugated antibody (Santa Cruz Biotechnology, Dallas, TX, USA), and HA-tagged proteases were detected using the anti-HA peroxidase-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA), according to manufacturers’ instructions.
3
Yeast Two-Hybrid and Co-IP Assays for Protein Interactions
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For Y2H, the vectors shown in Table S7 were transformed into Saccharomyces cerevisiae AH109 (Clontech). Transformations with empty vectors were used as negative controls. For co‐IP assays, pGWB605‐VlsPLA2WT or pGWB605‐VlsPLA2∆NLS1NLS2 was transiently co‐expressed with pGWB614‐NbVAMPA1 in N. benthamiana and pulled down as described above for the MS/MS assays. GFP‐tagged proteins were detected using a B2 anti‐GFP horseradish peroxidase‐conjugated antibody (Santa Cruz Biotechnology), and HA‐tagged proteases were detected using an anti‐HA peroxidase‐conjugated antibody (Sigma) according to the manufacturer's instructions.
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Western Blot Analysis of Parasitic Infection
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Protein lysates were prepared in radioimmunopreciptation assay (RIPA) buffer after harvesting protein from infected fibroblasts cultures in T25 flasks 24 hours post-infection, or from 5μm filter purified extracellular parasites. Laemmli sample buffer was added to samples and boiled for 5–10 minutes before loading on SDS-PAGE 4–20% pre-cast gradient gels (TGX) and running gels for 1.5-2hrs at 100V. Transfer to PVDF membranes (Millipore) was performed in Towbin buffer (20% methanol, Tris/Glycine buffer) for 2 hours at 100V, and blocking in 5% BSA/TBST was performed overnight in 4°C. Membranes were labeled in 5% BSA/TBST with anti-HA peroxidase conjugated antibody (Sigma, 1:200–1:1000), mouse anti-SAG1(Thermo Fisher, 1:100), rabbit TgALD1 antibody (1:200), and anti-rabbit HRP antibodies (Thermo Fisher, 1:10000) followed by development of signal with West Pico Plus Chemiluminescent substrate, or by LiCor anti-rabbit 680 and LiCor anti-mouse 800 secondary antibodies. Images of labeled blots were collected with a Li-COR instrument (Odyssey Imaging System).
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Western Blot Analysis Protocol
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Western blot analysis was performed as previously described [33 (link)]. Anti-HA peroxidase-conjugated antibody (Sigma, St. Louis, MO, USA) was used in 1:1000 dilution.
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