In situ streptavidin-gold labeling was performed as previously described.37 (link) Briefly, axonemes isolated from the ida7-1;SNAP::IC140 strain were resuspended in 400 μl of HMEEK buffer (30 mM HEPES, 25 mM KCl, 5 mM MgSO4, 0.1 mM EDTA and 1 mM EGTA, pH 7.2) and divided into two 1.5-ml Eppendorf tubes (200 μl each). One microliter of 1 mM BG-biotin (New England Biolabs, Ipswich, MA, USA) was added to each tube and incubated overnight with gentle rotation at 4°C. To wash away unbound BG-biotin, 1 ml of HMEEK was added to the tube and centrifuged (10,000 ×g for 2 min, 4°C). The wash step was repeated three times, and biotin-axonemes were resuspended in 200 μl of fresh HMEEK buffer. 5 μl of 80 μg/ml 1.4-nm-sized streptavidin nanogold particles (Nanoprobes Inc, Yaphank, NY, USA) was added to one tube and incubated at 4°C for 4 h; no streptavidin-gold was added to the sample in the second tube, which served as control. The labeled axonemes were washed by centrifugation (10,000 ×g for 2 min, 4 °C) and resuspended in HMEEK buffer.
Bg biotin
Manufactured by New England Biolabs
Sourced in United States
BG-biotin is a synthetic compound used for labeling and detection in various biological applications. It consists of a benzylguanine (BG) group conjugated to a biotin moiety. The BG group enables covalent attachment to SNAP-tag fusion proteins, while the biotin component allows for subsequent detection or purification using streptavidin-based methods.
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2 protocols using bg biotin
1
In situ Streptavidin-Gold Labeling of Axonemes
2
Quantifying Bem1-Cdc24 Interaction Kinetics
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JD53 cells expressing either Nub-Bem1 or Nub-ha were transformed with the plasmids carrying the respective CRU fusions. Cells were grown in selective media and serial dilutions were spotted on either nonselective media or media lacking histidine and uracil and containing various Met concentrations and 50 µM CuSO4. Cells were grown for 2 d at 30°.
PBBem1-SNAP fusion proteins, PBCdc24 and PBCdc24(D833G), were expressed as 6His-tagged proteins in the E. coli strain BL21DE3. Purification was achieved by IMAC and optional size exclusion chromatography. All proteins were buffered in HBSEP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4) and binding affinities were measured by SPR using a Biacore X100 system (GE Healthcare), essentially as described elsewhere (Renz et al. 2013 ). Briefly, purified PBBem1-SNAP (ligand protein) was covalently labeled with BG-Biotin (New England Biolabs) by SNAP tag chemistry and captured on a CM5 SPR chip (GE Healthcare) that was previously coated with an anti-biotin antibody (US-Biologicals). For the determination of kinetic parameters, purified PBCdc24 analyte protein was prepared in suitable concentrations in HBSEP buffer. Kinetic constants were calculated with the Biacore X100 Evaluation Software (Version 1.1; GE Healthcare).
PBBem1-SNAP fusion proteins, PBCdc24 and PBCdc24(D833G), were expressed as 6His-tagged proteins in the E. coli strain BL21DE3. Purification was achieved by IMAC and optional size exclusion chromatography. All proteins were buffered in HBSEP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4) and binding affinities were measured by SPR using a Biacore X100 system (GE Healthcare), essentially as described elsewhere (Renz et al. 2013 ). Briefly, purified PBBem1-SNAP (ligand protein) was covalently labeled with BG-Biotin (New England Biolabs) by SNAP tag chemistry and captured on a CM5 SPR chip (GE Healthcare) that was previously coated with an anti-biotin antibody (US-Biologicals). For the determination of kinetic parameters, purified PBCdc24 analyte protein was prepared in suitable concentrations in HBSEP buffer. Kinetic constants were calculated with the Biacore X100 Evaluation Software (Version 1.1; GE Healthcare).
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