The cell extracts obtained by the procedure above were incubated with α-HA antibody (12CA5, Sigma) for 1.5 h at 4°C. This solution was then mixed with Dynabeads Protein A (Invitrogen), followed by incubation for 1.5 h at 4°C. Thereafter, the beads were washed 3 times with lysis buffer. 25 μl of TEV buffer (50 mM Tris–HCl (pH 8.0), 5 mM EDTA (pH 8.0), 1mM DTT) were added to the beads with or without adding 15 U TEV protease (T4455, Sigma) followed by overnight incubation at 30°C. α-HA antibody (12CA5, Sigma) was used as the primary antibody and α-mouse IgG-HRP (GE Healthcare) was used as the secondary antibody for protein detection.
α ha antibody
Manufactured by Merck Group
The α-HA antibody is a laboratory reagent used to detect and identify proteins tagged with the hemagglutinin (HA) epitope. It is a specific and sensitive tool for immunodetection and immunoprecipitation applications.
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Lab products found in correlation
Dynabeads protein a, by Thermo Fisher Scientific (2 mentions)
α mouse igg hrp, by GE Healthcare (2 mentions)
α tubulin antibody, by Merck Group (2 mentions)
Tev protease, by Merck Group (1 mentions)
Ab18123, by Abcam (1 mentions)
Multi bead shocker, by Yasui Kikai (1 mentions)
Complete mini edta free protease inhibitor, by Roche (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
α flag m2 antibody, by Merck Group (1 mentions)
α gfp antibody, by Santa Cruz Biotechnology (1 mentions)
Ab56416, by Abcam (1 mentions)
Anti flag antibody magnetic beads, by Merck Group (1 mentions)
Sp8 lsm confocal microscope, by Leica (1 mentions)
Las af software, by Leica (1 mentions)
α flag m2 affinity gel, by Merck Group (1 mentions)
6 protocols using α ha antibody
1
Immunoprecipitation and Western Blot
2
Protein Extraction and Western Blot Analysis
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The cells cultured as described above were centrifuged and washed twice with cold PBS followed by freezing using liquid nitrogen. The frozen cells were suspended with lysis buffer (50 mM Tris–HCl (pH 7.5), 1 mM EDTA (pH 8.0), 10% glycerol, 150 mM NaCl, 0.05% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, Complete protease inhibitor (Roche)) and cells were disrupted by 0.5 mm zirconia beads and Multibead Shocker (Yasuikikai, Japan). The supernatants were then collected by centrifugation and the protein concentration was measured. Equal amounts of SDS buffer were added to the solutions and heated at 100°C for 3 min. The resulting samples were resolved by standard SDS-PAGE on a 12% polyacrylamide gel and transferred to a PVDF membrane by Wet transfer system. α-Atf1 antibody (ab18123, abcam), α-HA antibody (12CA5, Sigma), and α-tubulin antibody (T6074, Sigma) were used as primary antibodies, and α-mouse IgG-HRP (GE Healthcare) and α-rat IgG-HRP (Santa Cruz) were used as the secondary antibody for protein detection.
3
Meiosis Protein Immunoprecipitation Protocol
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Cells (450 mL) were harvested after 3 hr of synchronised meiosis. Three independent cultures were prepared for each strain. Pellets were lysed in cryo-mill and the resulting powder was resuspended in 25 mL of lysis buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 0.01% NP40, 5 mM β-mercaptoethanol, AEBSF, Serva protease inhibitors, 1 mM NEM, 1× Complete Mini EDTA-Free protease inhibitors; Roche). The lysate was cleared by centrifugation at 10,000 rpm for 1 hr. The supernatant was incubated with 5 μL of α-HA antibody (Sigma-Aldrich, H6908) for 3 hr at 4°C followed by the addition of 25 μL magnetic Dynabeads Protein G (Invitrogen, 10,004D) pre-equilibrated with wash buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, 0.01% NP40, 1 mM β-mercaptoethanol). The samples were incubated overnight at 4°C. Next day, the samples were centrifuged at 2500 rpm for 1 min at 4°C and the beads were washed six times with 150 μL of 20 mM ammonium bicarbonate. Proteins were eluted with 75 μL of 2× SDS Laemmli buffer and boiled for 5 min at 95°C. The samples were analysed by Proteomics Core Facility (EMBL, Heidelberg).
4
Investigating mTOR Pathway Regulation
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We used an α-ISG15 antibody from Santa Cruz (F-9) at 1:200, an α-SQSTM1/p62 antibody from Abcam, UK (ab56416) at 1:1000. We used an α-LC3 antibody from MBL, Japan (M152-3, clone 4E12) at 1:1000 and an α-ACTIN antibody from Sigma, Saint Louis, MO (AC-15, A5441) at 1:5000. We used an α-mTOR antibody from Sigma (PA5-34663) at 2 µg/sample for immunoprecipitation, an α-mTOR antibody from Cell Signaling (7C10) at 1:1000, an α-tubulin antibody from Sigma (T6074) at 1:5000, an α-ubiquitin antibody from Cell Signaling (P4D1) at 1:1000, and an α-NEDD8 antibody from Cell Signaling (19E3) at 1:1000. We used an α-HA antibody from Sigma (H6908) at 1:1000 for immunoblotting, and an α-GFP antibody from Santa Cruz Biotechnology (sc-81045) at 1:1000. We used an α-FLAG® M2 antibody from Sigma (F3165) at 1:10,000, an α-HA tag antibody—ChIP Grade from Abcam, UK (ab9110) at 1:5000, and an α-alpha-tubulin antibody from Genetex (GT114) at 1:40,000. We used α-HA antibody magnetic beads from Pierce (88837), and anti-flag antibody magnetic beads from Sigma (M2 M8823 Millipore) for immunoprecipitation.
5
Localization of Cpl1-HA in U. maydis
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To localize Cpl1‐HA in budding cells and filamentous hyphae, U. maydis strains constitutively expressing Cpl1‐HA were suspended in 2% YEPSlight containing 0.1 mM 16‐hydroxy hexadecanoic acid at a final OD600 of 0.5 and sprayed onto Parafilm. The Parafilm was placed on top of wetted paper towels inside square Petri dishes and incubated at 28°C for 17 h. The Parafilm was washed with water and blocked with phosphate‐buffered saline (PBS) containing 3% (wt/vol) bovine serum albumen and then incubated in α‐HA antibody (Sigma; 1:1500 dilution) diluted in PBS and 3% wt/vol bovine serum albumen at 4°C overnight. The samples were washed with PBS and incubated with a goat anti‐mouse IgG secondary antibody conjugated with AF488 (Life Technologies; 1:1500 dilution) for 1 h at 4°C. After washing, the samples were analysed using an SP8 LSM confocal microscope equipped with a 100× objective (NA 1.4; Leica). Fluorophores were excited with a pulsed white light laser source at 488 nm. Photon emission was detected with a hybrid detector at the appropriate wavelength (495–530 nm). Images were processed with Leica LAS AF software.
6
Stress Response Protein Interactions
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