Western blots were carried out as previously described26 (link). Primary antibodies used were anti-HA (Sigma, H3663-200UL; 1:1000 dilution), anti-FLAG (Sigma, F1804-200UG, 1:2000 dilution) and secondary antibody Goat anti-Mouse IgG1, HRP from Thermo Fisher Scientific, PA1 74421, (1:10000 dilution). Full scan uncropped Western blots are provided (Source Data 1). Immunolocalization of RAP2.3 was carried as described70 (link). Four-day-old seedlings containing the 35S:RAP2.33XHA transgene were treated for 3 h with 50 μM bortezomib or DMSO (control). Anti-HA primary antibody (Roche, Lewes, UK) and Alexa-Fluor-488-coupled anti rat secondary antibody (1:200 dilution) (Molecular Probes, Carlsbad, CA) were used for detection. Seedlings were counter stained using Propidium Iodide and visualised using confocal microscopy.
Anti ha primary antibody
Manufactured by Roche
Sourced in United Kingdom, Switzerland
The Anti-HA primary antibody is a laboratory reagent used to detect the presence of the hemagglutinin (HA) tag in recombinant proteins. It specifically binds to the HA epitope, allowing for identification and analysis of HA-tagged proteins in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg1 hrp, by Thermo Fisher Scientific (1 mentions)
H3663 200ul, by Merck Group (1 mentions)
F1804 200ug, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti ha primary antibody
1
Western Blot and Immunolocalization of RAP2.3
2
Tagging and Cross-linking of Kainate Receptors
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The X-linking of HA-tagged kainate receptors (HA-LiGluK2, HA-LiGluK2Δ16 and HA-GluK2) was achieved by first incubating hippocampal neurons for 10 min with an excess of the anti-HA primary antibody (Roche) and subsequently with an appropriate secondary antibody for 10 min (Gerrow and Triller, 2014 (link), Heine et al., 2008 (link)). After washing, neurons were used for the recordings of uncaging synaptic currents (uEPSCs).
3
Yeast Protein Expression and Detection
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Yeast cells containing plasmids expressing the different versions of AtHal3 and ScHal3 were tested for integrity and expression levels of these proteins. For this purpose, S. cerevisiae BY4741 (wild type) and its isogenic derivative hal3Δ were transformed with the different constructs and cells were grown in 10 ml of synthetic minimal medium lacking uracil at 28 °C to A660 of ∼0.8. Protein extracts were prepared as described in34 (link), and forty μg of total protein were resolved by SDS-PAGE. For immunobloting, proteins were transferred to Immobilon-P membranes (Millipore), and HA-tagged proteins were immunodetected using anti-HA primary antibody from Roche (1:1000 dilution) overnight, followed by the incubation with peroxidase conjugated mouse secondary antibody (GE Heathcare,1:20000 dilution) for one hour. Immunoreactive proteins were detected with ECL Prime (GE Healthcare) or Westar ηCUltra 2.0 (Cyanagen) kits.
4
Western Blot Analysis of NtMYC2a
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Tobacco leaves (about 0.1 g) were collected and ground in liquid nitrogen. The total protein was extracted with 200 μL 2×loading buffer (0.125 M Tris-HCl, pH 6.8, 25% glycerin, 5% SDS, 2.5% β-mercaptoethanol, 0.1% bromophenol blue) and separated by a 12% SDS-polyacrylamide gel and transferred to the PVDF membrane. The block was performed using 5% skim milk at 4 °C overnight. For the detection of NtMYC2a protein, the anti-HA primary antibody (Roche, Basel, Switzerland) and the HRP-labeled goat anti-mouse secondary antibody (Affinity Biosciences, Cincinnati, OH, USA) were used in turn and the signal was captured by fluorescence image analysis system (Tanon 5200, Shanghai, China) with the detection substrate (Pierce® ECL Western Blotting Substrate, Thermo Scientific, Waltham, MA, USA). As the control, NtActin was detected by mouse anti-actin primary antibody (CoWin Biosciences, Taizhou, Jiangsu, China) and the above-described secondary antibody using the same visualization method.
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