E. coli core RNAP (α2ββ′ω) with a SNAP-tag on the C-terminus of β′ (Tetone et al., 2017 (link)) was labeled with SNAP-Surface 647 (New England Biolabs) as described (Mumm et al., 2020 (link)) to produce RNAP647. Labeling stoichiometry measured as in Mumm et al. (2020) (link) was 54%. RNAP488-PEG-biotin was prepared in the same way, except that BG-488-PEG-biotin was used and unreacted dye was removed using a 30 kDa MWCO centrifugal filter (Microcon MRCF0R030; EMD Millipore); labeled protein was subjected to two cycles in which the protein was concentrated ~4 fold and then re-diluted to its original volume. To prepare σ70RNAP647 or σ70RNAP488-PEG-biotin, labeled core RNAP was incubated with σ70 as described (Harden et al., 2016 (link)).
Snap surface 647
Manufactured by New England Biolabs
SNAP-Surface 647 is a synthetic fluorescent labeling agent designed for covalent attachment to SNAP-tag fusion proteins. It provides a convenient and reliable way to label SNAP-tag fusion proteins with a bright, photostable red fluorescent dye, enabling their detection and visualization in a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Clip surface 647, by New England Biolabs (3 mentions)
Amicon ultra 0.5 ml concentrator, by Merck Group (3 mentions)
Spectramax i3x, by Molecular Devices (2 mentions)
Snap lumi4 tb, by PerkinElmer (2 mentions)
Gfp rab7, by Thermo Fisher Scientific (2 mentions)
Nepa21 electroporator, by Nepa Gene (2 mentions)
Axio observer a1 microscope, by Zeiss (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Snap biotin, by New England Biolabs (1 mentions)
Oregon green, by Thermo Fisher Scientific (1 mentions)
Snap surface 549, by New England Biolabs (1 mentions)
Tmr 6 maleimide, by Thermo Fisher Scientific (1 mentions)
Cy5 mono maleimide, by GE Healthcare (1 mentions)
Puromycin, by Merck Group (1 mentions)
11 protocols using snap surface 647
1
E. coli RNAP Fluorescent Labeling
2
Protein Labeling Efficiency Calculation
3
Neurotrophic Factor Receptor Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells (ATCC, mycoplasma negative) were transfected with a combination of 0.125 µg trkB/trkA/trkC in pcDNA plasmid and 0.125 µg p75NTR (all gifts from Moses Chao, Addgene plasmid #24088, #24089, #24093, and #24091, respectively) using Lipofectamine 2000 (Invitrogen, 11668-019) in a medium containing DMEM (Gibco, 41966-029), 10% FBS, 1% penicillin/streptomycin. Before labeling, cells were incubated in serum-free medium for 1 h. Aliquot of 0.1 µM BDNFSNAP was coupled to 0.3 µM of SNAP-surface 647 (New England Biolabs, S9136S) for 1 h at 37 °C and applied onto cells for 15 min at 4 °C and imaged using a Zeiss AxioObserver A1 microscope.
DRG from TrkBCreERT2::RFP mice was collected in PBS and incubated in 1 mg/ml collagenase IV (Sigma-Aldrich, C5138) and 0.05% Trypsin (Gibco, 25300-054) for 25 min each at 37 °C. Cells were filtered and suspended in medium containing DMEM (Gibco, 41966-029), 10% heat inactivated fetal bovine serum (PAA, A15101), 0.8% glucose, and 100 U of penicillin/streptomycin (Gibco, 15140-122). Cells were plated on glass coverslips treated with poly-L-lysine and stored at 37 °C. An equimolar concentration of BDNFSNAP was coupled to a mixture of SNAP-Biotin (New England Biolabs, S9110S) and QD655 quantum dots (Invitrogen, Q10121MP) at 37 °C for 30 min. Cells were labeled with the above mixture for 5 min and imaged using a Zeiss AxioObserver A1 microscope.
DRG from TrkBCreERT2::RFP mice was collected in PBS and incubated in 1 mg/ml collagenase IV (Sigma-Aldrich, C5138) and 0.05% Trypsin (Gibco, 25300-054) for 25 min each at 37 °C. Cells were filtered and suspended in medium containing DMEM (Gibco, 41966-029), 10% heat inactivated fetal bovine serum (PAA, A15101), 0.8% glucose, and 100 U of penicillin/streptomycin (Gibco, 15140-122). Cells were plated on glass coverslips treated with poly-L-lysine and stored at 37 °C. An equimolar concentration of BDNFSNAP was coupled to a mixture of SNAP-Biotin (New England Biolabs, S9110S) and QD655 quantum dots (Invitrogen, Q10121MP) at 37 °C for 30 min. Cells were labeled with the above mixture for 5 min and imaged using a Zeiss AxioObserver A1 microscope.
4
TR-FRET Receptor Clustering Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells stably or transiently expressing SNAP-GLP-1R were dual-labeled with the TR-FRET probe SNAP-Lumi4-Tb (40 nM, Cisbio) and SNAP-Surface 647 (1 μM, New England Biolabs) for 60 min at room temperature. These concentrations were selected because they provided optimum signal intensity at both measurement wavelengths. Where relevant, MβCD treatments were performed after labeling. Cells were washed and placed in HBSS in a white plate for a 10-min baseline measurement at 37 °C using a Spectramax i3x (Molecular Devices) plate reader fitted with a TR-FRET filter set (λEx = 335 nm, λEm = 616 nm and 665 nm, delay 30 μs, integration time 400 μs). TR-FRET was sequentially measured after agonist addition. The ratio of fluorescent signals at both emission wavelengths (665/616) was considered indicative of clustering because it reflects both transient and stable interactions between receptor protomers occurring within the long fluorescence lifetime of the excited terbium cryptate. Dose-response curves were constructed from AUC obtained from kinetic traces at different agonist concentrations and fitted to 4-parameter logistic curves to calculate potency (log EC50) and efficacy (Emax) values.
5
Purification and Labeling of Actin-Associated Proteins
6
Protein Labeling Efficiency Calculation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CLIP and SNAP tagged proteins were labeled using CLIP Surface-647 and SNAP Surface-647 dyes (New England Bio Labs), respectively. Purified protein at a concentration of 2 mg/ mL was incubated with 10 mM β-mercaptoethanol and 4x molar excess of dye at room temperature for 5 min, and then on ice overnight. Unlabeled dye was removed through dialysis in a Mini Dialysis Kit, 8 kDa cut-off (Cytiva) for 4 hours at 4°C, in buffer containing 50 mM phosphate pH 8.0, 300 mM NaCl, 5% glycerol, 30% sucrose, 10 mM β-mercaptoethanol, followed by multiple rounds of concentration and dilution through an Amicon Ultra 0.5 mL concentrator (Sigma-Aldrich). Labeling efficiency was calculated by measuring absorbance at 280 nm and 650 nm (A280 and A650) and using extinction coefficients of protein and dye (ε280 and ε650 ), as
The labeling efficiency for the proteins used in TIRF assays were Alexa 647-labeled Kif4A: 47%; Alexa 647-labeled PRC1: 17% and Alexa 647-labeled EB3: 45%.
The labeling efficiency for the proteins used in TIRF assays were Alexa 647-labeled Kif4A: 47%; Alexa 647-labeled PRC1: 17% and Alexa 647-labeled EB3: 45%.
7
Enhancing Chimeric Antigen Receptor T-Cell Expression
8
Live-cell imaging of EGFR trafficking
9
Live-cell imaging of EGFR trafficking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were electroporated with SNAP-EGFR57 using the NEPA21 Electroporator (Nepa Gene Co., Ltd), and transduced with GFP-Rab7 (Life Technologies, C10588) two days prior to imaging. Cells expressing SNAP-EGFR58 and GFP-Rab7 were labeled with DiIC16TCO, DiIC16’TCO, or DMSO control for 5 minutes in DPBS with 1% BSA. The probe mixture was replaced with DMEM ph(−) and the cells were allowed to incubate for 15 minutes at 37°C. 1 μM SNAP-Surface-647 (NEB, S9136S) was added as per the manufacturers protocol. The cells were washed 3x with warm DPBS and incubated in DMEM ph(−) at 37°C for 20 minutes prior to imaging. 2 mL fresh DMEM ph(−) was added to the cells, and cells expressing both SNAP-EGFR and GFP-Rab7 were found. One frame was taken before addition of 100 ng/mL EGF in DMEM and all subsequent stacks were taken after the addition of growth factor.
10
Fluorescent Labeling of Purified Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CLIP and SNAP tagged proteins were labeled using CLIP Surface-647 and SNAP Surface-647 dyes (New England Bio Labs), respectively. Purified protein at a concentration of 2 mg/ mL was incubated with 10 mM β-mercaptoethanol and 4x molar excess of dye at room temperature for 5 min, and then on ice overnight. Unlabeled dye was removed through dialysis in a Mini Dialysis Kit, 8 kDa cut-off (Cytiva) for 4 hours at 4°C, in buffer containing 50 mM phosphate pH 8.0, 300 mM NaCl, 5% glycerol, 30% sucrose, 10 mM β-mercaptoethanol, followed by multiple rounds of concentration and dilution through an Amicon Ultra 0.5 mL concentrator (Sigma-Aldrich). Labeling efficiency was calculated by measuring absorbance at 280 nm and 650 nm (A280 and A650) and using extinction coefficients of protein and dye (ε280 and ε650 ), as % labeling efficiency = 100 x (A650 / ε650)/(A280 / ε280)
The labeling efficiency for the proteins used in TIRF assays were Alexa 647-labeled Kif4A: 47%; Alexa 647-labeled PRC1: 17% and Alexa 647-labeled EB3: 45%.
The labeling efficiency for the proteins used in TIRF assays were Alexa 647-labeled Kif4A: 47%; Alexa 647-labeled PRC1: 17% and Alexa 647-labeled EB3: 45%.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!