l azidohomoalanine aha, by Thermo Fisher Scientific - Product details

1

Quantifying MYCN protein turnover

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Cells treated with 30 μM CHX (EMD) were harvested at various times for Western analysis. Signal intensity was quantified by ImageStudioLite. After normalization to α-tubulin, Log₂ values were plotted exponentially in Excel. Half-life (t_1/2) was determined using the Excel function, t_1/2=ln(2)/λ (where λ is the decay constant).
Nascent protein was examined using Click-iT protein detection kit (Life Technology). Cells were incubated for 20 min in RB medium with methionine-free IMDM (Life Technology), followed by labeling with 50 μM of L-azidohomoalanine AHA (Life Technology) for 1h. The cells were lysed in RIPA (Millipore) with inhibitors as above for 10 min, and sonicated. The lysate was centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was incubated with 2 μg of MYCN antibody with rotation for 2 hours, incubated with 30 μl of Protein A/G beads at 4°C overnight, and washed as above. Azide-modified protein were labeled with TAMRA and purified following manufacture’s protocol, separated by electrophoresis, and detected by laser scanner (Biorad-FX Pro) or by Western blot using anti-TAMRA antibody (Life Technology).^{36 (link)}

Qi D.L, & Cobrinik D. (2016). MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation. Oncogene, 36(13), 1760-1769.

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2

Visualizing Surface Proteins and Glycans

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For visualization of newly-synthesized proteins on the cell surface, neurons were incubated in methionine-free media subsequent to TNR epitope blocking: DMEM (4.5 mg/ml glucose, lacking pyruvate, methionine, glutamine, and cysteine; Life Technologies, Carlsbad, CA, USA) supplemented with 50 μM L-azidohomoalanine (AHA; Life Technologies), 812 μM MgCl2, 6.5 mM HEPES, 260 μM cysteine, 1:50 B27 (Gibco, Life Technologies), and 1:100 GlutaMAX (Gibco, Life Technologies). For visualization of surface glycans, 50 μM Click-IT™ GalNAz and/or GlcNAz (#C33365, C33367, Thermo Fisher Scientific, USA) were diluted directly into the neurons’ media subsequent to blocking, and remained throughout the experiment. Live, copper-free click labeling of surface AHA or glycans was performed after TNR labeling using strain-promoted azide-alkyne cycloaddition (SPAAC)⁶⁸. Neurons were incubated with 1:1000 dibenzocyclooctyne (DBCO) conjugated to AlexaFluor647 (#CLK-1302, Jena Bioscience, Germany) diluted in Ca²⁺/Mg²⁺-free Tyrode’s solution with 1 mM EGTA (#67-42-5; Merck, Germany).

Dankovich T.M., Kaushik R., Olsthoorn L.H., Petersen G.C., Giro P.E., Kluever V., Agüi-Gonzalez P., Grewe K., Bao G., Beuermann S., Hadi H.A., Doeren J., Klöppner S., Cooper B.H., Dityatev A, & Rizzoli S.O. (2021). Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R. Nature Communications, 12, 7129.

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3

Click Labeling of Nascent Proteins

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For click labeling, neurons were incubated for 9 h in a methionine‐free DMEM (4.5 mg/ml glucose, lacking pyruvate, methionine, glutamine, and cysteine; Life Technologies, Carlsbad, CA, USA) supplemented with 50 μM l‐azidohomoalanine (AHA; Life Technologies), 812 μM MgCl₂, 6.5 mM HEPES, 260 μM cysteine, 1:50 B27 (Gibco, Life Technologies), and 1:100 GlutaMAX (Gibco, Life Technologies). Click labeling was performed after fixation with a commercial kit (Click‐iT Cell Reaction Buffer Kit; Invitrogen), and with 5 mM Chromeo494‐alkyne (Jena Bioscience, Jena, Germany).

Truckenbrodt S., Viplav A., Jähne S., Vogts A., Denker A., Wildhagen H., Fornasiero E.F, & Rizzoli S.O. (2018). Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission. The EMBO Journal, 37(15), e98044.

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4

Metabolic Labeling of ULBP2 Protein

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HCT116-ULBP2 cells treated with DMSO or 40 µM PRL3-I for 18 h were washed once with PBS and then starved in DMEM without methionine, cysteine, and glutamine for 1 h at 37 °C. A final concentration of 50 µM of L-Azidohomoalanine (AHA) (Life Technologies), 5 mM of L-cysteine (Sigma) and 1× GlutaMAX (Life Technologies) were added for 1 h additional incubation. Cells were washed and replaced with complete DMEM supplemented with 10% FBS. Cell lysates were collected at different time points for Click-it biotinylation according to the manufacturer’s instructions. Biotinylated proteins were isolated using streptavidin microbeads (Miltenyi) for Western blot analysis to detect the protein synthesis of ULBP2.

Leung W.H., Vong Q.P., Lin W., Bouck D., Wendt S., Sullivan E., Li Y., Bari R., Chen T, & Leung W. (2015). PRL-3 mediates the protein maturation of ULBP2 by regulating the tyrosine phosphorylation of HSP60. Journal of immunology (Baltimore, Md. : 1950), 194(6), 2930-2941.

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5

Quantifying MYCN protein turnover

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Cells treated with 30 μM CHX (EMD) were harvested at various times for Western analysis. Signal intensity was quantified by ImageStudioLite. After normalization to α-tubulin, Log₂ values were plotted exponentially in Excel. Half-life (t_1/2) was determined using the Excel function, t_1/2=ln(2)/λ (where λ is the decay constant).
Nascent protein was examined using Click-iT protein detection kit (Life Technology). Cells were incubated for 20 min in RB medium with methionine-free IMDM (Life Technology), followed by labeling with 50 μM of L-azidohomoalanine AHA (Life Technology) for 1h. The cells were lysed in RIPA (Millipore) with inhibitors as above for 10 min, and sonicated. The lysate was centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was incubated with 2 μg of MYCN antibody with rotation for 2 hours, incubated with 30 μl of Protein A/G beads at 4°C overnight, and washed as above. Azide-modified protein were labeled with TAMRA and purified following manufacture’s protocol, separated by electrophoresis, and detected by laser scanner (Biorad-FX Pro) or by Western blot using anti-TAMRA antibody (Life Technology).^{36 (link)}

Qi D.L, & Cobrinik D. (2016). MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation. Oncogene, 36(13), 1760-1769.

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6

Detection of Newly Synthesized Proteins

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Newly synthesized proteins were detected using the Click-IT method (Life Technologies), according to the manufacturer’s instructions. Briefly MOCK- or full-length STAT6-transfected U373MG cells or control siRNA- or STAT6 siRNA-transfected U87MG cells were exposed to hypoxia for 18 h. Cells were subsequently washed with PBS, incubated in methionine-free DMEM for 1 h, then pulsed with 50 μM of the methionine analog, L-azidohomoalanine (AHA) (Cat # C10102; Life Technologies), for 2 h, after which cells were harvested. AHA-incorporated proteins were labeled with biotin using a Click-iT Biotin Protein Analysis Detection Kit (Cat# C33372; Invitrogen). Biotin-labeled proteins were pulled down using a streptavidin-agarose bead slurry (Pierce, Rockford, IL, USA) and analyzed by immunoblotting using an anti-HIF-1α antibody.

Park S.J., Kim H., Kim S.H., Joe E.H, & Jou I. (2019). Epigenetic downregulation of STAT6 increases HIF-1α expression via mTOR/S6K/S6, leading to enhanced hypoxic viability of glioma cells. Acta Neuropathologica Communications, 7, 149.

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7

Visualizing Newly Synthesized Proteins in Xenopus Eye Explants

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Stage 24 Xenopus eye explant cultures were incubated in methionine-free 60% L15 media for 6 h prior to the experiments. l-Azidohomoalanine (AHA; Life Technologies cat. no. C10102) 200 µM concentration was added for 1 h, then washed with embryo media followed by PBS. Explants were fixed with 2% Pfa/7.5% sucrose in PBS for 20 min at RT, washed 3× with 3% BSA/PBS and permeabilized with 0.1% Triton X-100 in PBS, and the Click reaction was performed with Click-iT Alexa Fluor 488 detection reagent, according to manufacturer's instructions (Life Technologies). Culture dishes were mounted in Fluorosave and imaged.

Konopacki F.A., Wong H.H., Dwivedy A., Bellon A., Blower M.D, & Holt C.E. (2016). ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis. Open Biology, 6(4), 150218.

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8

Comprehensive Antibody Validation for Cell Lines

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LNCaP, V16-D and PC-3 cells were obtained from ATCC. The following primary antibodies were used: PARP (Cat. No. 9542), BAX (2D2) (Cat. No. 89477), β-Actin (Cat. No. 3700), BIOTIN (Cat. No. 5597), AR (Cat. No. 5153) and GAPDH (Cat. No. 2118) were from Cell Signaling; G3BP1 (Cat. No. 611127) was from BD Biosciences; YB-1 (Cat. No. ab76149), m6A (Cat No. ab151230) and G3BP1 (Cat. No. ab56574) were from Abcam; YTHDF3 (Cat. No. NBP2-94636) was from Novus; m6A (Cat. No. 202 003) was from Synaptic Systems; ERα (Cat. No. SAB4500810) and Vinculin (Cat. No. V9131) were from Sigma; Fluorescent secondary antibodies (mouse, Alexa Fluor 488/594; rabbit, Alexa Fluor 488/594), TRIzol, RPMI, FBS, RNAiMAX transfection reagent, Dynabeads M-280 Streptavidin, DMEM, FBS, Click-iT Protein Reaction Buffer Kit, biotin-alkyne and L-azidohomoalanine (AHA) were from Life Technologies; FluorSave was from Merck; siAR siRNA (5′-UCAAGGAACUCGAUCGUAUUU-3′) targeting full-length AR was from Dharmacon; siESR1 (Cat. No. sc-29305), siMETTL3 (Cat. No. sc-92172), siWTAP (Cat. No. sc-63224), siKIAA1429 (Cat. No. sc-77700), siMETTL14 (Cat. No. sc-89054), and siControl (Cat. No. sc-37007) siRNAs were from Santa Cruz Biotechnology.

Somasekharan S.P., Saxena N., Zhang F., Beraldi E., Huang J.N., Gentle C., Fazli L., Thi M., Sorensen P.H, & Gleave M. (2021). Regulation of AR mRNA translation in response to acute AR pathway inhibition. Nucleic Acids Research, 50(2), 1069-1091.

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9

Quantifying Nascent Protein Synthesis in Neuronal Cells

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Cortical neurons were transfected with expression vectors for GFP or GFP-tagged TDP-43^WT or TDP-CTF. After 24 hrs, cells were incubated in methionine-free DMEM (Thermo Fisher Scientific) with or without 40 μM anisomycin (Sigma-Aldrich) for 1 hr at 37°, followed by incubation with 100 μg/mL L-azidohomoalanine (AHA) (Thermo Fisher Scientific) for 5 mins. Cells were washed with 1×PBS and fixed by 4% PFA, permeabilized with 0.2% Triton X-100 for 5 mins and blocked with 3% BSA in 1×PBS for 30 mins. Click-iT reaction cocktail (50 μL per sample) was prepared right before the end of blocking using Click-iT assay kits and Alexa Fluor 647-conjugated alkyne (Thermo Fisher Scientific). Samples were incubated for 30 mins at RT and washed with 3% BSA in 1×PBS before mounting. The mean pixel intensity of AHA in the cell body was quantified with ImageJ software.

Chou C.C., Zhang Y., Umoh M.E., Vaughan S.W., Lorenzini I., Liu F., Sayegh M., Donlin-Asp P.G., Chen Y.H., Duong D.M., Seyfried N.T., Powers M.A., Kukar T., Hales C.M., Gearing M., Cairns N.J., Boylan K.B., Dickson D.W., Rademakers R., Zhang Y.J., Petrucelli L., Sattler R., Zarnescu D.C., Glass J.D, & Rossoll W. (2018). TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nature neuroscience, 21(2), 228-239.

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10

Quantifying Nascent Protein Synthesis in Neuronal Cells

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Cortical neurons were transfected with expression vectors for GFP or GFP-tagged TDP-43^WT or TDP-CTF. After 24 hrs, cells were incubated in methionine-free DMEM (Thermo Fisher Scientific) with or without 40 μM anisomycin (Sigma-Aldrich) for 1 hr at 37°, followed by incubation with 100 μg/mL L-azidohomoalanine (AHA) (Thermo Fisher Scientific) for 5 mins. Cells were washed with 1×PBS and fixed by 4% PFA, permeabilized with 0.2% Triton X-100 for 5 mins and blocked with 3% BSA in 1×PBS for 30 mins. Click-iT reaction cocktail (50 μL per sample) was prepared right before the end of blocking using Click-iT assay kits and Alexa Fluor 647-conjugated alkyne (Thermo Fisher Scientific). Samples were incubated for 30 mins at RT and washed with 3% BSA in 1×PBS before mounting. The mean pixel intensity of AHA in the cell body was quantified with ImageJ software.

Chou C.C., Zhang Y., Umoh M.E., Vaughan S.W., Lorenzini I., Liu F., Sayegh M., Donlin-Asp P.G., Chen Y.H., Duong D.M., Seyfried N.T., Powers M.A., Kukar T., Hales C.M., Gearing M., Cairns N.J., Boylan K.B., Dickson D.W., Rademakers R., Zhang Y.J., Petrucelli L., Sattler R., Zarnescu D.C., Glass J.D, & Rossoll W. (2018). TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nature neuroscience, 21(2), 228-239.

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L azidohomoalanine aha

Lab products found in correlation

22 protocols using l azidohomoalanine aha

Quantifying MYCN protein turnover

Visualizing Surface Proteins and Glycans

Click Labeling of Nascent Proteins

Metabolic Labeling of ULBP2 Protein

Quantifying MYCN protein turnover

Detection of Newly Synthesized Proteins

Visualizing Newly Synthesized Proteins in Xenopus Eye Explants

Comprehensive Antibody Validation for Cell Lines

Quantifying Nascent Protein Synthesis in Neuronal Cells

Quantifying Nascent Protein Synthesis in Neuronal Cells

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