HEK293T cells were transfected with the pCAGGS plasmids encoding HA-tagged VP35 and NP using the PEI reagent according to the manufacturer’s instructions. At 36 h after transfection, the cell lysates were prepared with cold lysate buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% glycerol, and 0.05% NP-40) containing EDTA-free protease inhibitors (Roche). The cell lysates were subsequently mixed with EZview Red Anti-HA Affinity Gel beads (Sigma) and incubated at 4 °C overnight with gentle rocking. After washing with lysate buffer, precipitated proteins were subjected to SDS-PAGE and Western blot analyses with a monoclonal anti-HA antibody (Abcam, ab1424) diluted at 1:5000 and rabbit antiserum to EBOV NP (FS0169) [25 (link)] diluted at 1:2000. The bound antibodies were visualized using Immobilon Western (Millipore). Band intensities were quantified using Amersham Imager 600 Analysis Software.
Ezview red anti ha affinity gel beads
Manufactured by Merck Group
EZview Red Anti-HA Affinity Gel beads are pre-made agarose beads coated with an anti-HA (hemagglutinin) antibody. They are designed for the rapid and efficient purification of HA-tagged proteins from cell lysates or other sample types.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western, by Merck Group (1 mentions)
Ab1424, by Abcam (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Anti ha high affinity antibody, by Roche (1 mentions)
Dykddddk tag antibody, by Cell Signaling Technology (1 mentions)
Goat anti rabbit hrp, by Bio-Rad (1 mentions)
Antibodies against ha tag, by Merck Group (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Sigma protease inhibitor cocktail, by Merck Group (1 mentions)
Udp glo glycosyltransferase assay, by Promega (1 mentions)
Udp glcnac, by Promega (1 mentions)
5 protocols using ezview red anti ha affinity gel beads
1
Immunoprecipitation of EBOV VP35 and NP
2
Purification and Detection of NT5C2 Proteins
3
Chromatin Protein Immunoprecipitation Protocol
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Protein extracts were prepared from 0–12 hr embryos as described (Elfring et al. 1998 (link)). 500 μg of protein extract was incubated with 25 μl EZ view Red anti-HA Affinity Gel beads (Sigma-Aldrich) in HEGN100 buffer (10 mM HEPES pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.05% Tween-20, 10% glycerol) with protease inhibitors (1 μg/ml each Chymostatin, Leupeptin, Pepstatin A, Aprotinin, and 100 μg/ml PMSF) for 3 hr at 4° on a rotator. Beads were collected at 8200 × g for 1 min at 4°, washed in five alternating washes of 10 volumes of HEGN100 and HEGN500 (10 mM HEPES pH 8.0, 1 mM EDTA, 500 mM NaCl, 0.05% Tween-20, 10% glycerol), and samples were eluted in 40 μl 2X SDS sample loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 0.2% bromophenol blue, 100 mM DTT, 20% glycerol) by boiling at 95° for 5 min. Western blotting was done following standard procedures with anti-CHD1 at 1:1000 (McDaniel et al. 2008 (link)) and anti-dRtf1 at 1:3000, with goat anti-rabbit-HRP at 1:10,000 (Bio-Rad), and were detected with Super Signal West Dura (Pierce). Four biological replicates were done.
4
Immunoprecipitation of HA-HBeAg
5
Immunoprecipitation and Assay of OGT Activity
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Plasmid for expression of pcDNA2.1HA-OGT was a gift from Gerald W. Hart (University of Georgia). EZview red anti-HA affinity gel beads (Sigma, cat# E6779) were twice washed with lysis buffer (20 mM HEPES, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM B-glycerophosphate, pH 7.4). After exposure to 10 µM Ang1–7 for 30 minutes, cells were harvested in 1 mL phosphate-buffered saline, spun at 1000g for 5 minutes at 4˚C, and cell pellet was resuspended in 500 µL lysis buffer plus inhibitors (1M DTT, 200 mM benzamidine, 200 mM sodium vanadate, Sigma protease inhibitor cocktail [#P8340]). Clarified cell lysate was added to the prepared anti-HA beads and incubated for 2 hours at 4˚C with gentle rocking. Lysate/bead mix was spun for 30 seconds at 8200g, unbound fraction aspirated, and bead pellet washed three times in 750 µL lysis buffer. OGT activity was assessed using a UDP-Glo glycosyltransferase assay (Promega, catalog #V6961). Bound OGT-HA fusion proteins were resuspended in 50 µL OGT transferase buffer (25 mM HEPES, pH 7.2, 10 mM MnCl2, 1 mM EDTA, and 1 mM PMSF) with 100 µM OGT peptide substrate (AnaSpec, Inc.) and 100 µM UDP-GlcNAc (Promega, catalog #V7071). After 1 hour at room temperature, reactions were quenched with UDP-Glo detection reagent and luminescence was measured using a SpectraMax M5 plate reader (Molecular Devices).
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