E. coli expressed GST-tagged human eIF1AX (known as eIF1A), or globular domain (GD, 25 ~114aa), or N-tail deletion (ND, 25~144aa) or C-tail deletion (CD, 1~114aa) were purified by GST-pull down assays (#88822, Thermo Scientific) with glutathione magnetic beads from pre-cleared cell lysates of HEK293 cells stably expressing HA-Ago2 in the presence of 10μg/ml Ribonuclease A treatment. Western blot assays were performed with anti-Ago2 monoclonal antibodies (Abcam, ab57113, 1:1000 dilution). For the Ago2 domain screening assays, E. coli expressed His-tagged L1, PAZ, sumo-MID, PIWI domains and Sumo, which were purified by Ni-NTA resin, were performed GST-pull down assays with E. coli expressed GST-eIF1A. Western blot assays were performed with anti-His monoclonal antibodies (Cell Signaling, #2366S, - 1:1000 dilution).
Anti his monoclonal antibody
Manufactured by Cell Signaling Technology
The Anti-His monoclonal antibody is a laboratory tool used to detect and purify recombinant proteins containing a histidine tag. It recognizes the histidine tag sequence, allowing for the identification and isolation of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti ago2 monoclonal antibodies, by Abcam (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Western blotting, by Beyotime (1 mentions)
Mg132, by Merck Group (1 mentions)
Anti flag monoclonal antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Liquid scintillant, by Thermo Fisher Scientific (1 mentions)
Anti gfp monoclonal antibody, by Cell Signaling Technology (1 mentions)
Anti srpk1 monoclonal antibody, by BD (1 mentions)
Epidermal growth factor (egf), by Roche (1 mentions)
Anti gst monoclonal antibody, by BioLegend (1 mentions)
Brij 35, by Thermo Fisher Scientific (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Rnase, by Thermo Fisher Scientific (1 mentions)
Phenix imaging film, by Thermo Fisher Scientific (1 mentions)
Ni resin, by Thermo Fisher Scientific (1 mentions)
Srpin340, by Merck Group (1 mentions)
Anti ha monoclonal antibody, by Cell Signaling Technology (1 mentions)
Protein g beads, by Cell Signaling Technology (1 mentions)
Instantblue, by Abcam (1 mentions)
6 protocols using anti his monoclonal antibody
1
eIF1A Interaction with Ago2 Domains
2
eIF1A Interaction with Ago2 Domains
3
Ubiquitylation Assay via Western Blot
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Proteins were extracted using the KGN cell lysis buffer and subjected to western blotting (Beyotime, Shanghai, China). Briefly, the cell lysates were centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatants were mixed with 5 μL of primary antibodies and 40 μL of protein A + G agarose suspension beads (Santa Cruz, Dallas, Texas, USA) to incubate overnight at 4 °C. The beads were rinsed with a lysis buffer the following day. Next, the samples were boiled in a loading buffer for 10 min at 100 °C before subjecting them to western blotting.
KGN cells were transfected using the His-Ub plasmid and then treated with 30 μM MG132, (Sigma-Aldrich) for 6 h. Subsequently, a portion of the lysates was incubated with anti-His monoclonal antibody (Cell signaling, Rabbit, 2365, 1:1000) and used for ubiquitylation experiments; bead-binding proteins and the rest of the lysates were analyzed with IB.
KGN cells were transfected using the His-Ub plasmid and then treated with 30 μM MG132, (Sigma-Aldrich) for 6 h. Subsequently, a portion of the lysates was incubated with anti-His monoclonal antibody (Cell signaling, Rabbit, 2365, 1:1000) and used for ubiquitylation experiments; bead-binding proteins and the rest of the lysates were analyzed with IB.
4
Co-immunoprecipitation and in vitro Ubiquitination Assays
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For the co-immunoprecipitation (co-IP) assays, A549 cells were transfected with Flag-tagged RPS27a or vector control, then lysed. Subsequently, 70% of the lysate was incubated with anti-Flag monoclonal antibody (Cell signaling technology, USA) or control IgG, and the remaining 30% of the lysate was analyzed with IB. In vitro ubiquitination experiments followed protocols from previous studies using the Ni2+-NTA purification method. The A549 cells were transfected with His-Ub plasmids after transfection of RPS27a-siRNA for 24 h, then treated with 40 μM MG132 for 6 h. Subsequently, 70% of the lysate was incubated anti-His monoclonal antibody (Cell signaling technology) and used for ubiquitination experiments with co-IP assays; the bead-bound proteins and the other 30% of the lysate were analyzed with IB [29 (link)].
5
Protein Separation and Immunoblot
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Protein samples were separated on a 10 % SDS-PAGE gel, then stained with Coomassie brilliant blue or transferred to a polyvinylidene fluoride membrane (Millipore). The membranes were blocked with 5 % skim milk and immunoblotted with an anti-His monoclonal antibody (Cell Signaling Technology, #2366).
6
Reagents for Protein Purification and Analysis
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