Anti-GFP nanobody (Chromotek, gt-250) was diluted in 0.2 M sodium bicarbonate solution at pH 8.2 to a final concentration of ~60 μM. Alexa Fluor 647 NHS Ester stock solution (1 mg/mL in DMSO) was added into diluted Anti-GFP nanobody to a final dye concentration of ~120 μM. The mixture of nanobody and dye was incubated for 1 h at 25 °C. Then, free dyes were removed from the solution using a Zeba spin desalting column (Thermo Scientific, 89882). As the extinction coefficients are known, the concentrations of purified nanobody and conjugated AF647 dye were determined using light absorption at 280 and 647 nm wavelengths, respectively. The degree of labeling (the average number of dye molecules per protein) was calculated to be 1.08.
Anti gfp nanobody
Manufactured by Proteintech
The Anti-GFP nanobody is a small, single-domain antibody fragment that specifically binds to the Green Fluorescent Protein (GFP). It is derived from the variable domain of a camelid heavy-chain antibody. The Anti-GFP nanobody can be used as a tool for the detection, purification, and manipulation of GFP-tagged proteins in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Elyra ps1 system, by Zeiss (2 mentions)
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (2 mentions)
Janelia fluor 549, by Bio-Techne (1 mentions)
Np 40, by Merck Group (1 mentions)
Magnetic beads, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Carl Roth (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Alexa fluor 647 nhs, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti tom20, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by BD (1 mentions)
Anti gfp, by Takara Bio (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti hrs, by Enzo Life Sciences (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Anti giantin, by Fortrea (1 mentions)
8 protocols using anti gfp nanobody
1
Fluorescent Labeling of Anti-GFP Nanobody
2
Combined smFISH and Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
smFISH combined with immunofluorescence was performed similarly to smFISH with slight modifications. N2 and DUP98 patr-1(sam50[patr-1::GFP::3xFLAG])II (Andralojc et al., 2017 (link)) embryos were harvested as above with the exception that they were resuspended in methanol, freeze cracked in liquid nitrogen for 1 min, and transferred to acetone after ∼5 min total in methanol. Embryos were then incubated in acetone for 25 min before proceeding to hybridization/immunofluorescence. smFISH was then performed as above with the exception that a final concentration of 2.37 µg/ml Janelia Fluor 549 (Tocris, 6147) conjugated anti-GFP nanobody (Chromotek, gt-250) was incubated with the embryos overnight in hybridization buffer.
3
Alexa Fluor 647 Conjugation of Anti-GFP Nanobody
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-GFP nanobody
(Chromotek,
gt-250) was diluted in 0.2 M sodium bicarbonate solution at pH 8.2
to a final concentration of ∼60 μM. Alexa Fluor 647 NHS
Ester stock solution (1 mg/mL in DMSO) was added into diluted anti-GFP
nanobody to a final dye concentration of ∼120 μM. The
mixture of nanobody and dye was incubated for 1 h at 25 °C. Then,
free dyes were removed from the solution using a Zeba spin desalting
column (Thermo Scientific, 89882). As the extinction coefficients
are known, the concentrations of purified nanobody and conjugated
AF647 dye were determined using light absorption at 280 and 647 nm
wavelengths, respectively. The degree of labeling (the average number
of dye molecules per protein) was calculated to be 1.08.
(Chromotek,
gt-250) was diluted in 0.2 M sodium bicarbonate solution at pH 8.2
to a final concentration of ∼60 μM. Alexa Fluor 647 NHS
Ester stock solution (1 mg/mL in DMSO) was added into diluted anti-GFP
nanobody to a final dye concentration of ∼120 μM. The
mixture of nanobody and dye was incubated for 1 h at 25 °C. Then,
free dyes were removed from the solution using a Zeba spin desalting
column (Thermo Scientific, 89882). As the extinction coefficients
are known, the concentrations of purified nanobody and conjugated
AF647 dye were determined using light absorption at 280 and 647 nm
wavelengths, respectively. The degree of labeling (the average number
of dye molecules per protein) was calculated to be 1.08.
4
Immunoprecipitation of CLN3-PSEN1 Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were transfected with constructs encoding HA-CLN3 and GFP-PSENEN or with HA-CLN3 and GFP. Twenty-four h post transfection, cells were lysed in 100 mM NaCl, 50 mM Tris-HCl, 1 mM CaCl2, 1 mM MgCl2, 0.5% NP40 (Sigma, 21–3277), 1 mM PMSF (Roth, 6367.1), pH 7.5, cell debris sedimented for 10 min at 16,000 g and 10% of each supernatant were taken as an expression control (input) and immunoprecipitation was performed using anti-GFP nanobody coupled to magnetic beads (ChromoTek, gtma-20) for 30 min at 4°C. After washing with 100 mM NaCl, 50 mM Tris-HCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% Triton X-100, 1 mM PMSF, pH 7.5 protein complexes were subjected to SDS-PAGE and immunoblotting.
5
Visualizing Virus-Like Particles with Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Band purified MVs diluted in 20 μl 1 mM Tris pH 9.0 were sonicated and vortexed prior to being placed on coverslips for 30 min. Bound virus was fixed with 4% formaldehyde. When required, samples were permeabilized for 30 min with 1% TritonX-100 in PBS and blocked with 5% BSA (Sigma), 1% FCS for 30 min and immune-stained with anti-GFP nanobody (Chromotek) conjugated in-house to AlexaFluor647-NHS (Invitrogen) in 5% BSA, PBS at 4°C overnight. Coverslips were washed three times with PBS prior to mounting. Both mounting and imaging by STORM and SIM and Virusmapper modelling have been described previously [74 (link)]. Correlative super-resolution light and electron microscopy imaging of MVs was performed as previously described [101 (link)].
6
Super-Resolution Microscopy of Vaccinia Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2.5 x 106 VACV particles (WR strain, EGFP-F18 in tk locus [21 (link)]) were diluted in 100 μl 1 mM TRIS, pH 8, sonicated for 3x 30 s and incubated on gridded #1.5 glass-bottom petri dishes (Zell-Kontakt GmbH) for 1 hour at room temperature and fixed with 4 % PFA in PBS for 15 min. Samples were quenched with 50 mM NH4Cl in PBS for 10 min, washed in PBS, and incubated in permeabilization/blocking buffer (1% Triton X-100, 5% BSA, 1% FBS in PBS) for 30 min. Samples were labelled in blocking/permeabilisation buffer overnight at 4 °C or 2 hours at room temperature with anti-GFP nanobodies (Chromotek), labelled in-house with Alexa Fluor 647 NHS-ester (Life Technologies) with a dye-to- protein ratio of approximately 1, as previously described [22 (link)]. Samples were then washed 3x with PBS, fixed in 4% PFA in PBS for 10 min, quenched with 50 mM NH4Cl in PBS for 10 min and washed in PBS.
VACV samples were imaged in STORM buffer on a Zeiss Elyra PS.1 system, using a 100x TIRF objective with additional 1.6x magnification (as above) for SIM, SRRF and SMLM acquisition (Sup. Fig. 5 ). Buffer was exchanged to PBS and STED images were acquired on a Leica SP8, re-localising the same region of interest based on the grid. SMLM data acquisition parameters were 30,000 frames at 100% laser power 647 nm illumination with 405 nm stimulation and an exposure time of 33 ms per frame.
VACV samples were imaged in STORM buffer on a Zeiss Elyra PS.1 system, using a 100x TIRF objective with additional 1.6x magnification (as above) for SIM, SRRF and SMLM acquisition (
7
Monoclonal and Polyclonal Antibody Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following monoclonal and polyclonal antibodies or nanobodies were used in this study: anti-E-cadherin (C-terminus: BD Transduction Laboratories, 61018; N-terminus: Genetex/Biozol GTX134997), anti-Tsg101 (Abcam, 4A10), anti-GFP (Takara, 632592), anti-GFP-nanobodies (Chromotek), anti-actin (BD Transduction Laboratories, 612656), anti-Hrs (Enzo, A-5; GeneTex, GTX89364), anti-GAPDH (Abcam, 6C5), anti-giantin (Covance, PRB-114C), anti-PDI (BD Transduction Laboratories, 610946), anti-TOM20 (Santa Cruz, Sc11415).
8
Super-Resolution Microscopy of Vaccinia Virus
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!