3 × flag peptide (apexbio)  (ApexBio)

Journal: Nature

Article Title: Rixosomal RNA degradation contributes to silencing of Polycomb target genes

doi: 10.1038/s41586-022-04598-0

Figure Lengend Snippet: a , Genomic tagging of endogenous NOL9 with 3×Flag. b , Experimental design for protein immunoprecipitation from a chromatin fraction. c , Western blots showing fractionation of HEK 293FT cells. d , Silver-stained gel of Flag immunoprecipitations from wild-type and Flag–NOL9-expressing HEK 293FT cells. e , Volcano plot displaying results of tandem-mass-tag mass spectrometry of proteins enriched in Flag immunoprecipitations from Flag–NOL9-expressing cells relative to untagged cells from two independent experiments. P values calculated by two-sided t -test. Subunits of the rixosome, PRC1, PCR2 and H3K9me3-associated HP1 proteins are highlighted. f , Immunoprecipitations (IP) showing the association of RING1B and EZH2 with Flag–NOL9 in HEK 293FT cells with or without benzonase treatment. IB, immunoblot. g , Immunoprecipitations showing the association of RING1B with rixosome subunits PELP1, TEX10, SENP3 and WDR18 in HEK 293FT cells with or without benzonase treatment. h , Volcano plot displaying mass spectrometry results of proteins enriched in Flag immunoprecipitations from cells expressing Flag–PHC2 relative to untagged cells from two independent experiments. P values calculated by two-sided t -test. Subunits of the rixosome, PRC1, PCR2 and selected other proteins are highlighted.

Article Snippet: Flag-tagged proteins were purified from the soluble chromatin fraction using magnetic beads (Sigma, M8823) and eluted with 3×Flag peptides (APExBIO, A6001) in elution buffer (20 mM Hepes-KOH, pH7.5, 100 mM KOAc, 5 mM Mg(OAc) 2 , 1 mM EDTA, 10% Glycerol).

Techniques: Immunoprecipitation, Western Blot, Fractionation, Staining, Expressing, Mass Spectrometry

Journal: Molecular cell

Article Title: MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

doi: 10.1016/j.molcel.2019.06.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 3 × FLAG tag peptide , APExBIO , A6001.

Techniques: Recombinant, Protease Inhibitor, FLAG-tag, ATP Bioluminescent Assay, Activity Assay, Western Blot, Autoradiography, Negative Control, Software

Journal: Nature chemical biology

Article Title: Harnessing the Anti-Cancer Natural Product Nimbolide for Targeted Protein Degradation

doi: 10.1038/s41589-019-0304-8

Figure Lengend Snippet: (a) Nimbolide targets an intrinsically disordered region within RNF114 as assessed by PONDR. (b) Route for synthesis of the alkyne-functionalized nimbolide probe. (c) Gel-based ABPP analysis of pure human RNF114 protein labeled with nimbolide probe. In the upper two panels, pure RNF114 protein was pre-incubated with DMSO vehicle or nimbolide (100 μM, 30 min) prior to labeling with the nimbolide probe (10 μM, 1 h) in PBS. In the lower two panels, pure wild-type and C8A mutant RNF114 protein were labeled with the nimbolide probe (10 μM, 1 h) in PBS with 1 mg/ml BSA. Full-length and replicate blots can be found in and . (d) Gel-based ABPP analysis of nimbolide competition against IA-alkyne (10 μM) or JNS27 (50 μM) labeling of pure RNF114 protein. Structures of IA-alkyne and JNS27 probes are shown with reactive moieties highlighted in red. Shown also is gel-based ABPP analysis of nimbolide (50 μM) competition against JNS27 labeling of wild-type and C8A mutant RNF114 protein. In these experiments, DMSO or nimbolide was pre-incubated for 30 min prior to probe labeling for 1 h. Full-length and replicate blots can be found in and . (e) Nimbolide-alkyne labeling of Flag-RNF114 in 231MFP cells. 231MFP cells stably expressing a Flag-tagged RNF114 were treated with DMSO vehicle or nimbolide-alkyne for 2 h. RNF114 was subsequently enriched from harvested cell lysates and then rhodamine-azide was appended onto probe-labeled proteins by CuAAC, after which nimbolide-alkyne labeling was visualized by SDS/PAGE and in-gel fluorescence. Full-length and replicate blots can be found in . (f) Nimbolide-alkyne labeling of endogenous RNF114 in 231MFP cells. 231MFP cells were treated with DMSO vehicle or nimbolide-alkyne (50 μM) for 1.5 h. Biotin-azide was appended to probe-labeled proteins by CuAAC, and these proteins were subsequently avidin-enriched. Resulting pulled down proteins were analyzed by SDS/PAGE and Western blotting for RNF114. Full-length and replicate blots can be found in Gels shown in (c-f) are representative gels from n=3 biologically independent samples/group.

Article Snippet: Proteins were eluted using 2 50 μL washes of PBS supplemented with 250 ng/μL 3 × FLAG peptide (ApexBio A6001).

Techniques: Labeling, Incubation, Mutagenesis, Stable Transfection, Expressing, SDS Page, Fluorescence, Avidin-Biotin Assay, Western Blot