Search results for: Histone Deacelytase 4, Biotin conjugates
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Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling.
Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl-CoA (Ac-CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry-based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac-CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl-CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub-acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne-biotin Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne-TAMRA for in-gel visualization Support Protocol 1: Expression and purification of HAT mutants Support Protocol 2: Synthesis of Ac-CoA surrogates Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates Basic Protocol 3: Chemoproteomic identification of HAT substrates Basic Protocol 4: Validation of specific HAT substrates with western blotting.Jiabao Song, Zhen Han, Y George Zheng
1560 related Products with: Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling.
25 g100ug100 µg96 assays 1 kit10reactions 10 g50 ug 10 mg50 ug10reactions
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#32244728 2020/04/01
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Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin.
The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.Renjie Liao, Thai Pham, Diego Mastroeni, Paul D Coleman, Joshua Labaer, Jia Guo
2984 related Products with: Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin.
100ug Lyophilized1 mg100ug Lyophilized200.1 mg Protein A100 ug6 x 100 µg 1 mg5 mg100ug Lyophilized
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Approach to profile proteins that recognize post-translationally modified histone "tails".
Post-translational modifications (PTMs) of histones, proteins onto which DNA is packaged, are involved in many biological processes, including transcription, recombination, and chromosome segregation. As these PTMs can be dynamic, combinatorial, and mediators of weak interactions, the comprehensive profiling of all proteins that recognize histone PTMs is a daunting task. Here we describe an approach to design probes that can be used to identify proteins that directly interact with modified histones. Protein structure was used to guide the introduction of a photo-cross-linker in the probe, so as to convert weak interactions into covalent linkages. The probe also included an alkyne group to facilitate click chemistry-mediated conjugation of reporter tags for the rapid and sensitive detection (via rhodamine) and affinity enrichment (via biotin) of labeled proteins. In particular, we developed and validated a probe that can selectively capture proteins that recognize trimethyled lysine-4 of histone H3 (H3K4me3) in whole proteomes. A complete profiling of H3K4Me3 binding proteins should shed new light on cellular processes regulated by this PTM.Xiang Li, Tarun M Kapoor
2178 related Products with: Approach to profile proteins that recognize post-translationally modified histone "tails".
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Synthesis and characterization of highly sensitive heparin probes for detection of heparin-binding proteins.
Three labeled heparin species were synthesized as probes for heparin-binding protein detection. Heparin conjugated with 5([4,6-dichlorotriazin-2-yl]amino)fluorescein can be iodinated to a high specific activity. This probe specifically detected 40 pg histone on a dot blot without affinity purification. Heparin biotinylated on its naturally occurring primary amino groups also detected known heparin-binding proteins in a specific manner. This probe detected lower amounts of collagen I and basic fibroblast growth factor on nitrocellulose membranes than did the iodinated probe, with comparable detection times. To create more attachment sites for biotin, we covalently attached amino groups to the hydroxyl groups of heparin using 3-bromopropylamine hydrobromide. After biotinylation, the amino-rich probe detected heparin-binding proteins at the same or higher sensitivity as the biotinylated native heparin probe, using 100-fold less probe and much shorter detection times. This method of labeling is generally applicable to other polysaccharides, and would be useful when the amount of ligand is limited. We show that these three probes detect essentially the same spectrum of proteins in detergent extract of smooth muscle cell plasma membrane, and expect them to be useful probes for detection of cell-surface heparin receptors.N A Stearns, S Prigent-Richard, D Letourneur, J J Castellot