Azide peg3 biotin conjugate

Manufactured by Merck Group

Sourced in United States

The Azide-PEG3-biotin conjugate is a chemical compound that contains an azide functional group, a polyethylene glycol (PEG) linker, and a biotin moiety. This product is commonly used in various biochemical and biotechnological applications that involve the detection, isolation, or immobilization of biomolecules.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Azide-PEG3-biotin (7 citations) Biotin azide (6 citations)

11 protocols using azide peg3 biotin conjugate

Analyzing BAX Binding to Liposomes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant BAX (10 mM) was incubated with liposomes containing 0%, 5%, or 10% (E)-hexadec-2-en-15-ynal (Cayman Chemical) in the presence of BIM SAHB_A2 (10 μM) or vehicle (1% DMSO) for 2 h at room temperature. A click-chemistry reaction was then performed by adding 0.8 mM of premixed 1:1 CuSO₄:Tris((1-benzyl-4-triazolyl)methyl)amine (TBTA) complex, followed by 125 mM Azide-PEG3-biotin conjugate (Aldrich) and 1 mM sodium ascorbate. The reaction was incubated for 1 h at room temperature with gentle mixing. High-capacity streptavidin agarose beads (30 μL) were washed three times in 1 mL of liposomal release assay buffer containing 1% CHAPS. The beads were blocked in 3% BSA in PBS for 1 h at 4C, and then washed 3 times. The click reaction (1 μM) was added to the beads and rotated for 1 h at 4C. The beads were sequentially washed 3 times with liposomal buffer containing 1% CHAPS and then 3 times with PBS. Bound protein was eluted from the beads by adding 10 mg/mL biotin in 10% SDS and heating to 95°C for 10 min. Samples were prepared by adding 3x LDS loading dye containing 1M DTT to the elution mixture, followed by electrophoresis on a 4%–12% Bis-Tris gel and BAX western analysis using the HRP-BAX 2D2 antibody (Santa Cruz).

Cohen D.T., Wales T.E., McHenry M.W., Engen J.R, & Walensky L.D. (2020). Site-Dependent Cysteine Lipidation Potentiates the Activation of Proapoptotic BAX. Cell reports, 30(10), 3229-3239.e6.

+ Open protocol

+ Expand

Analyzing BAX Binding to Liposomes

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cohen D.T., Wales T.E., McHenry M.W., Engen J.R, & Walensky L.D. (2020). Site-Dependent Cysteine Lipidation Potentiates the Activation of Proapoptotic BAX. Cell reports, 30(10), 3229-3239.e6.

+ Open protocol

+ Expand

Photocrosslinking of HDAC1 Interactors

Check if the same lab product or an alternative is used in the 5 most similar protocols

Recombinant HDAC1 (200 ng) in 10 µL whole cell lysis buffer (as per 1.4.) was pretreated with 100 mM SAHA (1) or S20 or DMSO control for 15 minutes and then treated with PRP 8 (10 mM) or DMSO control for 40-min. Samples were cooled on ice and irradiated with 366 nm light for 30 min then incubated with 0.1 mM azide-PEG3- biotin conjugate, Sigma Aldrich 762024 (2.5 mM stock in DMSO) in the presence of 1mM CuSO4, 0.1 mM TBTA, 1 mM TCEP for 90 minutes at room temperature. Samples were then diluted with loading buffer, separated by SDS-PAGE and transferred to nitrocellulose membrane (as per 1.7. below). Membrane was blocked and incubated with IRDye® 800CW streptavidin and labeled protein signal was recorded with Odyssey Sa scanner at 800 nm. Immunoblotting with HDAC specific antibodies was done as per 1.7.

Aboukhatwa S.M., Hanigan T.W., Taha T.Y., Neerasa J., Ranjan R., El-Bastawissy E.E., Elkersh M.A., El-Moselhy T.F., Frasor J., Mahmud N., McLachlan A, & Petukhov P.A. (2019). Structurally Diverse Histone Deacetylase Photoreactive Probes: Design, Synthesis, and Photolabeling Studies in Live Cells and Tissue. ChemMedChem, 14(11), 1096-1107.

+ Open protocol

+ Expand

Tracking Cholesterol Dynamics in Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

K-562 and MCF7 cells were incubated in lipid-free media (RPMI and DMEM, respectively) containing 10 μg/mL alkyne cholesterol (Avanti, 700143) for 16 h. Samples were washed once in PBS and nuclei obtained using the EZ Prep nuclei kit, then incubated with 50 μM Azide-PEG3-biotin conjugate (Sigma-Aldrich, 762024) was dissolved in prewarmed buffer A and added to samples. The click reaction was initiated via addition of 2 mM CuBF4 in 2% (vol/vol) acetonitrile, and the reaction was left to proceed at 43 °C for 30 min with gentle agitation. The nuclei were then washed in buffer A and immediately used for preparation of nuclear extract for immunoprecipitation (using streptavidin magnetic beads, Thermo Fisher, 88816), chromatin immunoprecipitation (Click-ChIP with streptavidin magnetic beads), or immunofluorescence (using Streptavidin Alexa Fluor 594 conjugate, Thermo Fisher, S32356).

Loats A.E., Carrera S., Fleming A.F., Roberts A.R., Sherrard A., Toska E., Moorhouse A.J., Medler K.F, & Roberts S.G. (2021). Cholesterol is required for transcriptional repression by BASP1. Proceedings of the National Academy of Sciences of the United States of America, 118(29), e2101671118.

+ Open protocol

+ Expand

Biotinylation and Pull-down of RNA

Check if the same lab product or an alternative is used in the 5 most similar protocols

RNA was extracted from
PropSeMet treated MOLM13 cells as described above. CuAAC reaction
was then performed in 20 mM HEPES (pH 7.5) on 7 μg of RNA, 20
μM azide-PEG3–biotin conjugate (Sigma-Aldrich), 5 mM
CuSO₄, 15 mM THPTA, and 5 mM NaAsc, to a final volume of
100 μL. After initiating the reaction with NaAsc, the mixture
was stirred at 37 °C for 30 min, followed by quenching of the
reaction with 6 mM EDTA. Biotinylated RNA was then precipitated with
EtOH and redissolved in 30 μL of nuclease-free water. Streptavidin
pull-down was carried out using streptavidin beads (Invitrogen) according
to the manufacturer’s instructions. RNA was then analyzed via
RT-qPCR as described above with abundance of each transcript in the
pulled-down RNA compared to corresponding input RNA.

Mikutis S., Gu M., Sendinc E., Hazemi M.E., Kiely-Collins H., Aspris D., Vassiliou G.S., Shi Y., Tzelepis K, & Bernardes G.J. (2020). meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation. ACS Central Science, 6(12), 2196-2208.

+ Open protocol

+ Expand

Metabolic Labeling and Click Chemistry

Check if the same lab product or an alternative is used in the 5 most similar protocols

K-562 cells were incubated in lipid free media containing 10 μg/mL myristic acid-alkyne (Avanti) for 16 h. Cells were then harvested and washed once in PBS and the nuclei isolated using the EZ Prep nuclei kit. The purified nuclei were then incubated with 50μM Azide-PEG3-biotin conjugate (Sigma-Aldrich) in Buffer A and added to samples. The click reaction was initiated via addition of 2mM CuBF4 in 2% (v/v) acetonitrile, and the reaction was left to proceed at 43°C for 30 min with rotation. The nuclei were then washed in 0.1M HEPES/KOH (pH 7.4) and immediately used for either preparation of nuclear extract for immunoprecipitation, chromatin immunoprecipitation or immunofluorescence.

Moorhouse A.J., Loats A.E., Medler K.F, & Roberts S.G. (2022). The BASP1 transcriptional corepressor modifies chromatin through lipid-dependent and lipid-independent mechanisms. iScience, 25(8), 104796.

+ Open protocol

+ Expand

Efficient Protocol for Bioconjugation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

DMSO, NGF, Tris (2-carboxyethyl) phosphine (TCEP), tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methylamine (TBTA), Cy5-azide and Azide-PEG3-biotin Conjugate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). CuSO₄ was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Pierce streptavidin magnetic beads and zeba spin desalting columns were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Cheng L., Wang Y., Xiang L, & Qi J. (2021). Heat Shock Cognate 70 kDa Protein Is the Target of Tetradecyl 2,3-Dihydroxybenzoate for Neuritogenic Effect in PC12 Cells. Biomedicines, 9(10), 1483.

+ Open protocol

+ Expand

Click Chemistry Reagents for Bioconjugation

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEPES (Sigma-Aldrich, 99.5% purity, copper ≤ 5 ppm); MgCl₂.H₂O (Sigma-Aldrich, 99% purity, copper ≤ 5 ppm); LiOH·H₂O (Sigma-Aldrich, ≥ 99% purity, copper ≤ 5 ppm); sodium ascorbate (Sigma-Aldrich, 98% purity); NaCl (Sigma-Aldrich, 99% purity); KCl (ACP Chemicals, 99%); sodium acetate (Bio Basic, 99% purity); 5-hexyn-1-ol (Sigma-Aldrich, 96% purity); propargyl alcohol (Sigma-Aldrich, 99% purity); 3-azido-7-hydroxycoumarin (AK Scientific 98% purity); azide-PEG3-biotin conjugate (Sigma-Aldrich, purity not specified by the manufacturer); NaOH (Sigma-Aldrich, 99% purity); CuSO₄ (Fisher Scientific, 99% purity); Azido-PEG2-NHS ester (BroadPharm, 98% purity); Fluorescein-NHS ester (BroadPharm, 95% purity); AFDye 546 (Click Chemistry Tools, ≥95%); Lysozyme, from chicken egg white (Sigma-Aldrich, ≥ 98% purity); Nuclease P1 (NEB, 100,000 Units/mL).

Liu K., Lat P.K., Yu H.Z, & Sen D. (2020). CLICK-17, a DNA enzyme that harnesses ultra-low concentrations of either Cu+ or Cu2+ to catalyze the azide-alkyne ‘click’ reaction in water. Nucleic Acids Research, 48(13), 7356-7370.

+ Open protocol

+ Expand

Biotin-mediated Protein Interactome Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols

DOPAC, laccase from Rhus vernicifera, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), bis(4-nitrophenyl) phosphate (BNPP) and azide-PEG₃-biotin conjugate were obtained from Sigma Aldrich (St. Louis, USA). p-Toluensulfonic acid monohydrate (PTSA), n-butanol, toluene, copper (II) sulfate pentahydrate, protease inhibitor cocktail and Chemi-Lumi One Super were purchased from nacalai tasque (Kyoto, Japan). 2-Propyl-1-ol was obtained from Tokyo Chemical Industry (Tokyo, Japan). Streptavidin, HRP conjugate was purchased from Funakoshi (Tokyo, Japan). Anti-actin antibody, anti-aryl hydrocarbon receptor (AhR) antibody, anti-Keap1 antibody, horseradish peroxidase-linked anti-mouse IgG and horseradish peroxidase-linked anti-goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Streptavidin Mag Sepharose was purchased from GE health care (Little Chalfont, UK). All other chemicals such as benzyl azide were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Nakashima S., Liu Z., Yamaguchi Y., Saiki S., Munemasa S., Nakamura T., Murata Y, & Nakamura Y. (2016). A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite. Biochemistry and Biophysics Reports, 7, 240-245.

+ Open protocol

+ Expand

Mapping Cholesterol Localization in Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

K-562 and MCF7 cells were incubated in lipid free media (RPMI and DMEM, respectively) containing 10μg/ml alkyne-Cholesterol (Avanti) for 16 hrs. Samples were washed once in PBS and nuclei obtained using the EZ Prep nuclei kit then incubated with 50μM Azide-PEG3-biotin conjugate (Sigma-Aldrich) was dissolved in prewarmed Buffer A and added to samples.. The click reaction was initiated via addition of 2mM CuBF4 in 2% (v/v) acetonitrile, and the reaction was left to proceed at 43 o C for 30 mins with gentle agitation. The nuclei were then washed in Buffer A and immediately used for either preparation of nuclear extract for immunoprecipitation (using streptavidin magnetic beads, ThermoFisher), chromatin immunoprecipitation (Click-ChIP with streptavidin magnetic beads) or immunofluorescence (using Streptavidin Alexa Fluor 594 conjugate, ThermoFisher).

Loats A.E., Carrera S., Fleming A.F., Roberts A.R.E., Sherrard A., Toska E., Medler K.F., & Roberts S.G.E. (2021). Nuclear cholesterol is required for transcriptional repression by BASP1.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!