Cells were grown to ~80% confluency at the time of assay. Complete medium was replaced by AHA medium formulated with DMEM, high glucose, no glutamine, no methionine, no cystine (Gibco), 10% dialyzed FBS (Gibco), 1% GlutaMAX (Gibco), 1% Penicillin-Streptomycin (Gibco), 62.6 mg/L L-Cysteine (Sigma), 250uM L-Azidohomoalanine (AHA, Click Chemistry Tools). For non-AHA control, the AHA was replaced by 30 mg/L L-methionine (Sigma). For Anisomycin (ANS) treatment, AHA medium was further added with 10 μg/mL Anisomycin (Cell Signaling). After 3 h of incubation, cells were scraped in cold DPBS, washed twice with cold DPBS and lysed in RIPA buffer. Protein concentration of the lysate was quantified by BCA and input protein concentrations were normalized to 2.1 mg/mL for downstream click chemistry. 50μL of the normalized cell lysate was used for click reaction with biotin-Alkyne (Click Chemistry Tools) by using Click-&-Go Click Chemistry Reaction Buffer Kit (Click Chemistry Tools). The reaction mixture was then methanol precipitated and was resuspended in 2xLaemmi buffer (BIO-RAD) with 2-mercaptoethanol (BIO-RAD) and denatured at 95°C for 10 min. Final protein concentration is 1 mg/mL. The sample was then resolved by SDS–PAGE, and total protein signal was detected by One-Step Blue Protein Gel Stain (Biotium). The biotin signal was detected by HRP-Conjugated Streptavidin (Thermo Scientific).
L azidohomoalanine
Manufactured by Vector Laboratories
Sourced in United States
L-Azidohomoalanine is a non-canonical amino acid that can be used for the metabolic labeling of proteins. It is a structural analog of the natural amino acid methionine and can be incorporated into newly synthesized proteins in place of methionine.
Automatically generated - may contain errors
Lab products found in correlation
Biotin alkyne, by Vector Laboratories (3 mentions)
Methionine, by Thermo Fisher Scientific (2 mentions)
Cystine, by Thermo Fisher Scientific (2 mentions)
One step blue protein gel stain, by Biotium (2 mentions)
Anisomycin, by Cell Signaling Technology (2 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
Dialyzed fbs, by Thermo Fisher Scientific (2 mentions)
Click go click chemistry reaction buffer kit, by Vector Laboratories (2 mentions)
L methionine, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
L cysteine, by Merck Group (2 mentions)
Azido palmitic acid 1346, by Vector Laboratories (2 mentions)
Dlm 2062, by Cambridge Isotopes (2 mentions)
Algal amino acid mixture u 13c, by Cambridge Isotopes (2 mentions)
2 deoxy d glucose u 13c6, by Cambridge Isotopes (2 mentions)
Penicillin streptomycin 1514, by Thermo Fisher Scientific (2 mentions)
Dmem medium without l methionine, by Thermo Fisher Scientific (2 mentions)
L cysteine and l glutamine 21013, by Thermo Fisher Scientific (2 mentions)
Proteinase k eo0491, by Thermo Fisher Scientific (2 mentions)
7 protocols using l azidohomoalanine
1
Measuring Newly Synthesized Proteins
2
Metabolic Labeling of Newly Synthesized Proteins
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24 hours post transfection, cells in 10 cm dishes were prepared for metabolic labeling to examine newly synthesized proteins. Cells were washed twice with warm PBS. DMEM containing only high glucose was added to cells to deplete endogenous methionine and cysteine for 45 minutes. Next, the medium was replaced with DMEM containing 10% dialyzed FBS, 2% L-glutamine, .2 mM L-cysteine, 25 mM HEPES, 1 mM sodium pyruvate, penicillin (25,000 U/ml) and streptomycin (25,000 μg/ml), and 4 mM L-azidohomoalanine (Click Chemistry Tools) for 4 hours. After this incubation period, cell lysates were collect in RIPA buffer and prepared for immunoprecipitation.
3
Exosome Labeling with Fluorescent Tags
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L-azidohomoalanine (AHA), tetraacetylated N-azidoacetyl-D-mannosamine (ManNAz), and DBCO-Cy3 were purchased from Click Chemistry Tools (San Diego, CA, USA) and used as received. DBCO-PEG4-Biotin used for biotinylation of exosome was obtained from Sigma-Aldrich (St. Louis, MO). The nanoparticle sizes were measured using Brookhaven Zetaplus (Holtsville, NY), TEM of exosomes were imaged using a Tecnai FEG TEM (FEI Tecnai 12 Spirit Biotwin, FEI Company, Hillsboro, OR).
4
Multiomics Analysis of Cell Lines
5
Measuring Newly Synthesized Proteins
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Cells were grown to ~80% confluency at the time of assay. Complete medium was replaced by AHA medium formulated with DMEM, high glucose, no glutamine, no methionine, no cystine (Gibco), 10% dialyzed FBS (Gibco), 1% GlutaMAX (Gibco), 1% Penicillin-Streptomycin (Gibco), 62.6 mg/L L-Cysteine (Sigma), 250uM L-Azidohomoalanine (AHA, Click Chemistry Tools). For non-AHA control, the AHA was replaced by 30 mg/L L-methionine (Sigma). For Anisomycin (ANS) treatment, AHA medium was further added with 10 μg/mL Anisomycin (Cell Signaling). After 3 h of incubation, cells were scraped in cold DPBS, washed twice with cold DPBS and lysed in RIPA buffer. Protein concentration of the lysate was quantified by BCA and input protein concentrations were normalized to 2.1 mg/mL for downstream click chemistry. 50μL of the normalized cell lysate was used for click reaction with biotin-Alkyne (Click Chemistry Tools) by using Click-&-Go Click Chemistry Reaction Buffer Kit (Click Chemistry Tools). The reaction mixture was then methanol precipitated and was resuspended in 2xLaemmi buffer (BIO-RAD) with 2-mercaptoethanol (BIO-RAD) and denatured at 95°C for 10 min. Final protein concentration is 1 mg/mL. The sample was then resolved by SDS–PAGE, and total protein signal was detected by One-Step Blue Protein Gel Stain (Biotium). The biotin signal was detected by HRP-Conjugated Streptavidin (Thermo Scientific).
6
Multiomics Analysis of Cell Lines
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All cell lines were purchased from ATCC including HeLa (ATCC CCL-2), Raw264.7 (ATCC TIB-71), MDA-MB-231 (ATCC HTB-26), MDA-MB-468(ATCC HTB-132), MCF7 (ATCC HTB-22), 3T3-L1 MBX (ATCC CRL-3242), U-87 MG (ATCC HTB-14). Azido-palmitic acid (1346) and L-Azidohomoalanine (1066) were purchased from Click chemistry tools. D-glucose (1,2,3,4,5,6,6-D7, 97–98%, DLM-2062), algal amino acid mixture (U-13C, 97–99%, CLM-1548), 2-deoxy-D-glucose (U-13C6, 99%, CLM-10466) were purchased from Cambridge isotope laboratories. Deuterium oxide (151882), Taxol (T7402) and Gemcitabine (G6423) were purchased from Sigma-Aldrich. DMEM medium (11965), FBS (10082), penicillin/streptomycin (1514), DMEM medium without L-methionine, L-cysteine and L-glutamine (21013), DMEM without glucose (11966) and proteinase K (EO0491) were purchased from ThermoFisher Scientific. CaF2 substrates (CAFP25–1, CAFP13–1 and CAFP-76–26-1U) were purchased from Crystran.
7
Quantifying ZEB1 Nascent Translation
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