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Biotin alkyne

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Biotin-Alkyne is a small molecule that contains a biotin moiety and an alkyne functional group. It can be used for the detection and labeling of biomolecules that possess reactive alkyne groups.

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Lab products found in correlation

Thpta, by Merck Group (3 mentions) L azidohomoalanine, by Vector Laboratories (3 mentions) Biotin peg4 alkyne, by Vector Laboratories (2 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) Click go protein reaction buffer kit, by Vector Laboratories (2 mentions) Ascorbic acid, by Thermo Fisher Scientific (1 mentions) L aha, by Vector Laboratories (1 mentions) Horseradish peroxidase conjugated streptavidin, by Proteintech (1 mentions) Np40 lysis buffer, by Thermo Fisher Scientific (1 mentions) Irdye 800cw streptavidin, by LI COR (1 mentions)

8 protocols using biotin alkyne

1

Methionine Analog Incorporation Assay

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Infected RNVC were serum-starved for 48 h and were cultured in methionine-free medium for 1 h prior to performing the assay. The culture medium was then replaced with medium containing 25 µM AHA (Click Chemistry Tools) for 2 h to allow the incorporation of this methionine analog into newly synthesized proteins. Cells were collected in RIPA lysis buffer and equal amount of proteins were labeled with biotin-alkyne according to the manufacturer’s protocol (Click Chemistry Tools). Briefly, the following reagents were added to the protein samples at the specified final concentration: 20 µM biotin-alkyne (Click Chemistry Tools), 2 mM CuSO4 (Bio Basic), 15 mM ascorbic acid (Fisher Bioreagents), and 5 mM THPTA (Sigma-Aldrich). The samples were incubated for 30 min in the dark and were then boiled in Laemmli buffer for 5 min. Equal amounts of proteins were loaded on SDS-PAGE gels and were analyzed for total biotinylation levels by Western blotting using a streptavidin-HRP conjugate.
Giguère H., Dumont A.A., Berthiaume J., Oliveira V., Laberge G, & Auger-Messier M. (2018). ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression. Scientific Reports, 8, 13605.
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2

Enrichment and Identification of O-GlcNAcylated Proteins

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For enrichment and identification of the O-GlcNAcylated proteins, experiments were performed based on the procedure of Woo and co-workers49 . Briefly, the proteins in PBS containing 1% SDS were diluted with PBS and incubated with 100 μM THPTA (Sigma-Aldrich, 762342), 0.5 mM CuSO4, 2.5 mM fresh sodium ascorbate and 200 μM either Biotin-PEG4-Alkyne (Click Chemistry Tools, TA105) for immunoblotting or Biotin-Alkyne probe for proteomics at 37 °C for 4 h.
For biotin-immunoprecipitation, proteins were precipitated and resuspended into 100 μL PBS containing 1% SDS. The protein solutions were diluted with PBS to lower the final concentration of SDS into 0.2% and incubated with pre-washed 40 μL streptavidin beads slurry at room temperature for 2 h with gentle rotation. Beads were washed sequentially with 0.2 % SDS/PBS three times and PBS three times. Enriched proteins were eluted with SDS sample buffer and subjected to SDS-PAGE.
Ge Y., Ramirez D.H., Yang B., D’Souza A.K., Aonbangkhen C., Wong S, & Woo C.M. (2021). Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nature chemical biology, 17(5), 593-600.
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3

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).
Ni C., Schmitz D.A., Lee J., Pawłowski K., Wu J, & Buszczak M. (2022). Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly. Cell reports, 38(13), 110597.
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4

Quantifying Newly Synthesized Proteins

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Ten million cells were washed and grown in methionine-free media for 30 min to deplete methionine stores and then supplemented by l-azidohomoalanine (l-AHA) (50 μM final concentration for 4 h). Cell lysates were measured for OD at 260 and equal protein amounts were subjected to click chemistry using biotin alkyne (Click Chemistry Tools) as per manufacturer’s instructions. Total protein was precipitated with trichloroacetic acid, suspended in suspension buffer [8 M urea in 50 mM Tris–HCl (pH 7.4), 1 mM DTT] and biotin-tagged protein was isolated using streptavidin MyOne C1 magnetic beads (Thermo Fisher). Beads were washed three times with 8 M urea in Tris–HCl (pH 7.4), boiled for 10 min in SDS buffer and supernatant was subjected to western blotting.
Karmakar S., Ramirez O., Paul K.V., Gupta A.K., Kumari V., Botti V., de los Mozos I.R., Neuenkirchen N., Ross R.J., Karanicolas J., Neugebauer K.M, & Pillai M.M. (2022). Integrative genome-wide analysis reveals EIF3A as a key downstream regulator of translational repressor protein Musashi 2 (MSI2). NAR Cancer, 4(2), zcac015.
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5

Enrichment and Identification of O-GlcNAcylated Proteins

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For enrichment and identification of the O-GlcNAcylated proteins, experiments were performed based on the procedure of Woo and co-workers49 . Briefly, the proteins in PBS containing 1% SDS were diluted with PBS and incubated with 100 μM THPTA (Sigma-Aldrich, 762342), 0.5 mM CuSO4, 2.5 mM fresh sodium ascorbate and 200 μM either Biotin-PEG4-Alkyne (Click Chemistry Tools, TA105) for immunoblotting or Biotin-Alkyne probe for proteomics at 37 °C for 4 h.
For biotin-immunoprecipitation, proteins were precipitated and resuspended into 100 μL PBS containing 1% SDS. The protein solutions were diluted with PBS to lower the final concentration of SDS into 0.2% and incubated with pre-washed 40 μL streptavidin beads slurry at room temperature for 2 h with gentle rotation. Beads were washed sequentially with 0.2 % SDS/PBS three times and PBS three times. Enriched proteins were eluted with SDS sample buffer and subjected to SDS-PAGE.
Ge Y., Ramirez D.H., Yang B., D’Souza A.K., Aonbangkhen C., Wong S, & Woo C.M. (2021). Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase. Nature chemical biology, 17(5), 593-600.
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6

MKRN3 Regulation of Protein Synthesis

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300,000 cells were seeded and transduced with MKRN3 and control lentivirus. 60 h later, the medium was replaced with methionine-free medium (Thermo Fisher Scientific; #A1451701) for 2 h. Cells were incubated with 50 µM L-AHA (Clickchemistrytools; #1066-25) for 8 h and harvested in lysis buffer. L-AHA was detected using click chemistry with 50 µM Biotin Alkyne (Clickchemistrytools; #1266–5) with the Click-&-Go Protein Reaction Buffer Kit (Clickchemistrytools; #1262) per the manufacturer’s protocol. The extracted protein was detected by Western blotting as described above. The blots were probed with a horseradish peroxidase–conjugated streptavidin (Proteintech; #SA0001) and developed with Enhanced Chemiluminescence (GE; #RPN2232).
Li K., Zheng X., Tang H., Zang Y.S., Zeng C., Liu X., Shen Y., Pang Y., Wang S., Xie F., Lu X., Luo Y., Li Z., Bi W., Jia X., Huang T., Wei R., Huang K., Chen Z., Zhu Q., He Y., Zhang M., Gu Z., Xiao Y., Zhang X., Fletcher J.A, & Wang Y. (2021). E3 ligase MKRN3 is a tumor suppressor regulating PABPC1 ubiquitination in non–small cell lung cancer. The Journal of Experimental Medicine, 218(8), e20210151.
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7

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).
Ni C., Schmitz D.A., Lee J., Pawłowski K., Wu J, & Buszczak M. (2022). Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly. Cell reports, 38(13), 110597.
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8

Quantifying ZEB1 Nascent Translation

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One day after seeding 300, 000 cells/well, cells were infected with AdSLFN12 or AdCMV. Sixty hours later, the medium was replaced with methionine-free medium (Thermo Fisher, #21013024) for two hours. Cells were incubated with 50μM L-Azidohomoalanine (AHA) (Clickchemistrytools, Scottsdale, AZ, #106625) for 8 hours and lysed in NP-40 lysis buffer (Thermo Fisher, #FNN0021) with 1%SDS. AHA was detected using click chemistry with 50μM Biotin Alkyne (Clickchemistrytools, #1266–5) with Click-&-Go™ Protein Reaction Buffer Kit (Clickchemistrytools, #1262) per manufacturer’s protocol. After click chemistry, ZEB1 was immunoprecipitated using rabbit anti-ZEB1 and protein-A magnetic beads (Bio-Rad Laboratories, #161–4013). Proteins were resolved by 10% SDS-PAGE, transferred to 0.45μm nitrocellulose membrane, and blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE, #927–50010). Biotin was labeled with IRDye 800CW Streptavidin per manufacturer’s protocol (LI-COR, #926–32230) and ZEB1 was labeled with IRDye 680LT Donkey anti-Rabbit IgG Secondary Antibody. Images were acquired using a LI-COR-Clx and analyzed using Image Studio (LI-COR).
Al-Marsoummi S., Vomhof-DeKrey E, & Basson M.D. (2019). Schlafen12 Reduces the Aggressiveness of Triple Negative Breast Cancer through Post-Transcriptional Regulation of ZEB1 That Drives Stem Cell Differentiation. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 53(6), 999-1014.
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