Thpta

Manufactured by Vector Laboratories

Sourced in United Kingdom, United States

THPTA is a reagent used in bioconjugation applications. It functions as a coupling agent to facilitate the covalent attachment of biomolecules, such as proteins or peptides, to other molecules or surfaces.

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Lab products found in correlation

Biotin azide, by Vector Laboratories (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Sodium ascorbate, by Merck Group (1 mentions) Pharos fx plus molecular imager, by Bio-Rad (1 mentions) Azido rhodamine, by Vector Laboratories (1 mentions) Prolong glass, by Thermo Fisher Scientific (1 mentions) Alexa fluor 647 azide, by Vector Laboratories (1 mentions) Irdye 800cw streptavidin, by LI COR (1 mentions) Alamarblue hs reagent, by Thermo Fisher Scientific (1 mentions) Odyssey blocking buffer tbs, by LI COR (1 mentions) High capacity streptavidin agarose resin, by Thermo Fisher Scientific (1 mentions) Zeba spin desalting columns 7k mwco 0.5 ml, by Thermo Fisher Scientific (1 mentions) Revert total protein stain kit, by LI COR (1 mentions) Sh sy5y neuroblastoma cells, by American Type Culture Collection (1 mentions) 0.2 μm nitrocellulose, by Bio-Rad (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions) Imagej, by Leica camera (1 mentions) Azdye 488 azide, by Vector Laboratories (1 mentions) Stellaris 5, by Leica camera (1 mentions) Cuso4, by Sinopharm (1 mentions)

8 protocols using thpta

Azide-Alkyne Click Reaction for Labeling

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Cells fixed after HPG uptake were permeabilized with PBS containing 1% BSA/0.1% Saponin for 15 min and washed 2x in Click buffer (100 mM Tris-HCl, pH 7.4) before the addition of Click reaction mix. The reaction mix was made by sequential addition of 10 mM Sodium Ascorbate (Sigma, A7631), 2 mM THPTA (Click Chemistry Tools, 1010), 0.5 µM AFdye488 azide plus (Click Chemistry Tools, 1475) and 1x click buffer to CuSO₄ (0.5 mM final conc., [Sigma, 209198]). Samples were incubated for 30 min in the dark at room temperature. Cells were washed with FACS buffer and surface staining was performed as normal.

Heieis G.A., Patente T.A., Almeida L., Vrieling F., Tak T., Perona-Wright G., Maizels R.M., Stienstra R, & Everts B. (2023). Metabolic heterogeneity of tissue-resident macrophages in homeostasis and during helminth infection. Nature Communications, 14, 5627.

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Azido-rhodamine Labeling of Automodified Proteins

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The autoMARylation reactions were stopped by methanol:chloroform precipitation with subsequent collection of the precipitates by centrifugation. The protein pellets were dissolved and ‘clicked’ to azido-rhodamine (Click Chemistry Tools) in Denaturing CC Buffer (100 mM HEPES pH 8.0, 4 M urea, 0.5 M NaCl, 2% CHAPS, 100 μM azido-rhodamine, 5 mM THPTA [Click Chemistry Tools], 1 mM CuSO₄, 5 mM sodium ascorbate) in the dark at room temperature for 2 hr. The clicked proteins were collected by methanol:chloroform precipitation with centrifugation, dissolved in 1x SDS Loading Buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and incubated at 100°C for 10 min in the dark. The clicked proteins were run on a 10% PAGE-SDS gel in the dark. After washing twice with 10% methanol and twice with MilliQ H₂O, the gel was then visualized on a Bio-Rad Pharos FX Plus Molecular Imager (excitation: 532 nm, emission: 605 nm) (Gibson and Kraus, 2017 (link); Gibson et al., 2016 (link)).

Palavalli Parsons L.H., Challa S., Gibson B.A., Nandu T., Stokes M.S., Huang D., Lea J.S, & Kraus W.L. (2021). Identification of PARP-7 substrates reveals a role for MARylation in microtubule control in ovarian cancer cells. eLife, 10, e60481.

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Synthesis and Characterization of DA^yne and Its Interaction with PDIA3

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All chemicals required for the synthesis of DA^yne, preparation of buffers, mushroom tyrosinase (Lot#: SLBZ0022), NBT, Amicon ultra 0.5 mL centrifugal filters (3k) and click reactions were purchased from Sigma-Aldrich unless otherwise stated. NMR solvents were obtained from Cambridge Isotope Laboratories Inc. SH-SY5Y neuroblastoma cells were acquired from ATCC. Cell culture media, additives, and consumables were purchased from Corning. High capacity streptavidin agarose resin, Zeba Spin Desalting Columns (7K MWCO, 0.5 mL), PDIA3 (ERp57) antibody (Cat. no. CL2444), and DAPI were obtained from Thermo Scientific. alamarBlue HS reagent and ProLong Glass were purchased from Invitrogen. BCA kits and C-18 spin columns were bought from Pierce. Mini-PROTEAN TGX Precast Gels, 0.2 μm nitrocellulose, and filter paper transfer stacks were obtained from BioRad. Biotin azide, THPTA, Alexa Fluor 647 azide, and azide agarose beads were purchased from Click Chemistry Tools. REVERT total protein stain kit, Odyssey TBS blocking buffer, and IRDye800CW streptavidin were purchased from LI-COR. Recombinant PDIA3 and PDI inhibitor screening kit (fluorometric) were obtained from BioVision (Cat. no. 7601–100 and K840–100, respectively).

Hurben A.K., Erber L.N., Tretyakova N.Y, & Doran T.M. (2021). Proteome-Wide Profiling of Cellular Targets Modified by Dopamine Metabolites Using a Bio-Orthogonally Functionalized Catecholamine. ACS chemical biology, 16(11), 2581-2594.

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Visualizing HSV-1 Genome Dynamics

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U2OS cells were inoculated with EdC-labeled HSV-1 (MOI = 50) for 1 h at 4°C in presence of cycloheximide (100 μg/mL). Then the cells were washed twice with PBS, and incubated in infection medium with cycloheximide (100 μg/mL) at 37°C for 3 h. The samples were fixed immediately with 4% (w/v) PFA in PBS for 10 min and permeabilized with 1% Triton X-100 for 5 min at room temperature. Viral genomes were visualized using copper(I)-catalyzed azide alkyne cycloaddition as previously described.⁴⁷ (link) Briefly, the fixed samples were incubated with freshly prepared click chemistry staining mix (10 μM AZDye 488-azide [#1475-1, Click Chemistry Tools], 1 mM CuSO4 [#7758-99-8, Sinopharm Chemical Reagent Co., Shanghai], 10 mM sodium ascorbate [#S105026, Aladdin, Shanghai], 10 mM amino-guanidine [#A151036, Aladdin, Shanghai], and 1 mM THPTA [#760952-88-3, Click Chemistry Tools]). DAPI staining and slide mounting were performed as described in the immunofluorescence section. Images were acquired with a confocal microscope (Leica Stellaris 5) and analysis was performed with ImageJ (NIH).

Xie J., Wang S., Zhong Y., Gao M., Tian X., Zhang L., Pan D., Qin Q., Wu B., Lan K., Sun Z.J, & Zhang J. (2023). Oncolytic herpes simplex virus armed with a bacterial GBP1 degrader improves antitumor activity. Molecular Therapy Oncolytics, 29, 61-76.

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Membrane Protein Extraction and Analysis

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PrestoBlue^® Cell Viability Reagent, Mem-PER™ Plus Membrane Protein Extraction Kit, and all reagents for cell culture were purchased from Life Technologies (Carlsbad, CA, USA). Bradford Protein Assay and Clarity™ Western ECL Substrate were obtained from Bio-Rad (Hercules, CA, USA). Protease inhibitor cocktail and lovastatin were purchased from Sigma (Saint Louis, MO, USA). Primary antibodies against Rab11A and Rap1A/Rap1B were obtained from Abcam (Cambridge, UK) and primary antibodies against β-actin along with secondary HRP-linked antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
Iodoacetamide and WaLP were purchased from Sigma (Saint Louis, MO, USA), DTT, CuSO₄, TCEP from Fluorochem (Derbyshire, United Kingdom) and THPTA, biotin-PEG₃-azide from Click Chemistry Tools (Scottsdale, AZ, USA). Sequencing grade chymotrypsin was purchased from Promega (Madison, WI, USA).

Małolepsza J., Marchwicka A., Serwa R.A., Niinivehmas S.P., Pentikäinen O.T., Gendaszewska-Darmach E, & Błażewska K.M. (2022). Rational design, optimization, and biological evaluation of novel α-Phosphonopropionic acids as covalent inhibitors of Rab geranylgeranyl transferase. Journal of Enzyme Inhibition and Medicinal Chemistry, 37(1), 940-951.

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Celastrol Proteomic Profiling Protocol

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Celastrol (purity ≥98%) was purchased from Bethealth People Biomedical Technology (Beijing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan). N-Acetyl-l-cysteine and deferoxamine mesylate were obtained from AbMole BioScience (USA). Click chemistry reaction and LC–MS/MS reagents include: TAMRA-azide, Biotin-azide and THPTA (ClickChemistryTools, USA); NaVc and CuSO₄ (Sigma–Aldrich, USA); high capacity neutravidin agarose beads, TEAB and sequencing grade modified trypsin (Thermo, USA); Oasis HLB Extraction Cartridge (Waters, USA); Pierce™ Quantitative Fluorometric Peptide Assay Kit (Thermo, USA).
Specific primary antibodies anti-PRDX1, anti-PRDX2, anti-PRDX3, anti-PRDX4, anti-PRDX5, anti-PRDX6, anti-HO-1 (Abcam, UK), anti-Vimentin, anti-PPARγ, anti-COL1Al (Proteintech, China) and anti-β-actin (Affinity Biosciences, China) were used.

Luo P., Liu D., Zhang Q., Yang F., Wong Y.K., Xia F., Zhang J., Chen J., Tian Y., Yang C., Dai L., Shen H.M, & Wang J. (2021). Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1. Acta Pharmaceutica Sinica. B, 12(5), 2300-2314.

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RNA Biotinylation with Click Chemistry

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Typically, ∼50–100 μg of total RNA was used in the Click reaction. The Click reaction contained 10 mM Tris pH 7.5, 2 mM biotin azide (Click Chemistry Tools), 10 mM sodium ascorbate made fresh (Sigma), 2 mM THPTA (Click Chemistry Tools), and 100 μM copper sulfate. The reaction was incubated for 30 min in the dark at 25°C. The reaction was then cleaned up using a Quick RNA Mini kit (Zymo Research) for small scale biotinylation tests (<10 μg RNA), or through standard ethanol precipitation with sodium acetate for larger amounts of RNA (>10 μg). RNA was then eluted and resuspended to 1 μg/μl in 50 mM NaCl.

Engel K.L., Lo H.Y., Goering R., Li Y., Spitale R.C, & Taliaferro J.M. (2021). Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq. Nucleic Acids Research, 50(4), e24.

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Click Chemistry Labeling of K562 Cells

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Ac₄ManNAz labeled and unlabeled K562 cells were collected and washed with PBS. Cells were then resuspended in 320 μL of cold PBS containing 50 μM probe 3 and 200 μg/mL BSA. CuAAC catalyst buffer was prepared by sequentially adding of CuSO₄ (Aladdin), THPTA (Click Chemistry Tools), aminoguanidine (Aladdin) and sodium ascorbate (Aladdin) to PBS in a 1:5:20:50 molar ratio, and placed on ice for 10 min before adding to the cells. Then 10 μL of different concentrations of catalyst buffer were added to the cell suspension to make final concentrations of 50 μM, 100 μM and 150 μM of copper ions, and incubated at 4 °C for 15 min. Cells were washed with PBS and incubated with streptavidin-cy3 (1:5000) in staining buffer (PBS containing 0.2% BSA and 0.1% sodium azide, BD Pharmingen) for 30 min at 4 °C. The labeled cells were washed with PBS and analyzed with a FACSAria SORP flow cytometer (BD Biosciences). Data were analyzed using FlowJo (version 10.8.1) software.

Zheng J., Zheng Z., Fu C., Weng Y., He A., Ye X., Gao W, & Tian R. (2023). Deciphering intercellular signaling complexes by interaction-guided chemical proteomics. Nature Communications, 14, 4138.

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