Cell lysate or protein extracts obtained from sucrose gradient fractions were supplemented with Pierce Lane Marker Reducing Sample Buffer (Thermo Scientific #39000), heated at 95°C for 10 min, and separated by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes, then membranes were blocked overnight at 4°C in 5% milk prepared in 1× Tris-Buffered Saline (TBS) − 0.1% Tween20 supplemented with dibenzocyclooctyne-PEG4-biotin conjugate (Sigma-Aldrich #760749; 50 µM final concentration). Membranes were washed three times in 1× TBS − 0.1% Tween20 for 10 min each, then incubated with Precision Protein StrepTactin-HRP Conjugate (BioRad #1610380; 1:1000 in 5% milk prepared in 1× TBS − 0.1% Tween20) for 1 hr at room temperature, then washed again. Membranes were subsequently developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare #RPN2236). Images were acquired through the ChemiDoc MP Imaging System. ImageJ software (v 1.45s) was used for quantitation of AHA signal intensities.
Dibenzocyclooctyne peg4 biotin conjugate
Manufactured by Merck Group
Sourced in United States
The Dibenzocyclooctyne-PEG4-biotin conjugate is a chemical compound used in various biomedical research applications. It consists of a dibenzocyclooctyne moiety, a polyethylene glycol (PEG4) linker, and a biotin functional group. This compound can be utilized in click chemistry reactions and other biomolecular labeling techniques.
Automatically generated - may contain errors
Lab products found in correlation
Precision protein streptactin hrp conjugate, by Bio-Rad (1 mentions)
Pierce lane marker reducing sample buffer, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Lysyl endopeptidase lys c, by Fujifilm (1 mentions)
Dbco amine, by Merck Group (1 mentions)
Nhs activated sepharose fast flow, by GE Healthcare (1 mentions)
Af488 azide, by Jena Biosciences (1 mentions)
Trypsin gold, by Promega (1 mentions)
Benzonase, by Merck Group (1 mentions)
2 protocols using dibenzocyclooctyne peg4 biotin conjugate
1
Quantitative Western Blot Assay for AHA
2
Purification and Labeling of Ribosome and Cas9
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Purified E. coli ribosome
(E. coli B strain) was purchased from New England
Biolabs (MA, USA) and diluted with dilution buffer (50 mM HEPES, 50
mM KCl, pH 7.5, 10 mM MgAc2) to a concentration of 1 mg/mL.
Purified recombinant Cas9 from S. pyogenes fused
with a Halo-tag was generated in house, as described by Deng et al.25 (link) DSBSO was synthesized similarly as described
by Burke et al.24 (link) For enrichment similar
as described by Kaake et al.15 (link) (BARAC Method),
Dibenzocyclooctyne-PEG4-biotin conjugate (#760749-5MG, Sigma-Aldrich)
was used without further purification. Biotin was pulled using Pierce
High Capacity Streptavidin Resin (# 20359, Thermo). DBCO beads were
synthesized in house: NHS-activated Sepharose fast flow (#17-0906-01,
GE Healthcare) was incubated to varying concentrations of dibenzocyclooctyne-amine
(DBCO-amine, #761540, Sigma-Aldrich). The prepared beads were stored
as 50% slurry in a 1:1 ethanol:water mixture. AF488-Azide (#CLK-1275-1,
Jena Bioscience) was used to test success of bead–DBCO coupling.
Trypsin gold was purchased from Promega (Mannheim, Germany) and lysyl
endopeptidase (LysC) was from Wako (Neuss, Germany). Benzonase—pharmaceutical
production purity—was from Merck (Darmstadt, Germany).
(E. coli B strain) was purchased from New England
Biolabs (MA, USA) and diluted with dilution buffer (50 mM HEPES, 50
mM KCl, pH 7.5, 10 mM MgAc2) to a concentration of 1 mg/mL.
Purified recombinant Cas9 from S. pyogenes fused
with a Halo-tag was generated in house, as described by Deng et al.25 (link) DSBSO was synthesized similarly as described
by Burke et al.24 (link) For enrichment similar
as described by Kaake et al.15 (link) (BARAC Method),
Dibenzocyclooctyne-PEG4-biotin conjugate (#760749-5MG, Sigma-Aldrich)
was used without further purification. Biotin was pulled using Pierce
High Capacity Streptavidin Resin (# 20359, Thermo). DBCO beads were
synthesized in house: NHS-activated Sepharose fast flow (#17-0906-01,
GE Healthcare) was incubated to varying concentrations of dibenzocyclooctyne-amine
(DBCO-amine, #761540, Sigma-Aldrich). The prepared beads were stored
as 50% slurry in a 1:1 ethanol:water mixture. AF488-Azide (#CLK-1275-1,
Jena Bioscience) was used to test success of bead–DBCO coupling.
Trypsin gold was purchased from Promega (Mannheim, Germany) and lysyl
endopeptidase (LysC) was from Wako (Neuss, Germany). Benzonase—pharmaceutical
production purity—was from Merck (Darmstadt, Germany).
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