HEK293T cells were grown on laminin‐coated glass coverslips. Cells were co‐transfected with 100 ng SNAP‐LGR5 or SNAP‐FZD5 and 150 ng myc‐NEDD4, HA‐NEDD4‐CS, myc‐NEDD4L, HA‐NEDD4L‐CA or pcDNA4 as a control with Fugene according to the manufacturer's instructions. After 24 h of transfection, cells were labelled with 1 μM SNAP‐Surface Alexa 488 (NEB) for 15 min at 4°C to block endocytosis. Cells were then immediately washed with fresh RPMI media and fixed in 4% paraformaldehyde. Cells were incubated with primary antibodies NEDD4 (Santa Cruz) or NEDD4L (Cell Signaling) for 1 h at RT followed by a secondary antibody conjugated to Alexa‐568 (Invitrogen) and DAPI (Sigma) in 2% BSA‐PBS (Roche). Cells were mounted in Prolong Diamond (Life technologies) and imaged using a DeltaVision Core system.
Snap surface alexa 488
Manufactured by New England Biolabs
SNAP-Surface Alexa 488 is a fluorescent labeling reagent designed for covalent attachment to SNAP-tag fusion proteins. It provides a rapid and specific way to label SNAP-tag fusion proteins with the Alexa Fluor 488 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 568, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Nedd4l, by Cell Signaling Technology (1 mentions)
Bsa pbs, by Roche (1 mentions)
Nedd4, by Santa Cruz Biotechnology (1 mentions)
2 protocols using snap surface alexa 488
1
SNAP-LGR5 and SNAP-FZD5 Colocalization
2
Immunofluorescence Staining Protocol
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Transfected cells on coverslips were fixed with 4% paraformaldehyde in PBS solution for 15 min, followed by three 5 min washes in PBS. Permeabilization was performed on all cells with 0.5% TritonX100 in PBS solution for 3 min, before three more 5 min washes in PBS. Cells were blocked with 3% BSA in PBS for 1 h at room temperature before staining with primary antibodies at appropriate dilutions in 3% BSA overnight at 4˚C. Corresponding fluorescent secondary antibodies in 3% BSA were used to stain the sample cells for 1 h at room temperature after primaries were washed off. Where SNAP-tagged proteins were being visualized, SNAPligand TMR-Star (NEB) or SNAP-surface-Alexa488 (NEB) were added at the secondary antibody staining stage. Following staining and washing, cells were mounted upside-down on microscopy slides in MOWIOL, allowed to cure for >12 h at 4˚C, and imaged on a Zeiss LSM880 Airyscan/AiryscanFast confocal microscope with a 20x air objective.
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