HeLa cells were seeded at 1 × 104 per well in μClear 96 well plates (Greiner). Next day, the cells were transfected with plasmids coding for GFP-Miro1 or a GFP-tagged non-related protein. 24 h post transfection, the cells were washed twice with PBS and fixed with 4% (w/v) paraformaldehyde (PFA) in PBS for 10 min at RT and blocked with 5% BSA in TBST for 30 min at RT. Incubation with purified Miro1 Nbs or AF647 conjugated Nbs (100–200 nM in 5% BSA in TBST) or rabbit anti-Miro1 antibody (# NBP1-89011, Novus Biologicals) was performed overnight at 4°C. Unbound nanobodies were removed by three washing steps with TBST. Unlabelled Miro1 Nbs were detected by addition of a Cy5-conjugated Goat Anti-Alpaca IgG (Jackson Immunoresearch) according to manufacturer’s guidelines. For Miro1 antibody detection, an AF647 conjugated goat anti-rabbit secondary antibody (Invitrogen) was used. Nuclei were subsequently stained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and images were acquired immediately afterwards with an ImageXpress™ Micro Confocal High Content Screening system (Molecular Devices) at 40x magnification.
Af647 conjugated goat anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
The AF647 conjugated goat anti-rabbit secondary antibody is a laboratory reagent designed for use in immunoassays and other applications. It is a polyclonal antibody that binds to rabbit primary antibodies and is conjugated to the Alexa Fluor 647 fluorescent dye. This product can be used to detect and visualize rabbit primary antibodies in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Nbp1 89011, by Novus Biologicals (2 mentions)
Cy5 conjugated goat anti alpaca igg, by Jackson ImmunoResearch (2 mentions)
Imagexpress micro confocal high content screening system, by Molecular Devices (2 mentions)
μclear 96 well plate, by Greiner (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
2 protocols using af647 conjugated goat anti rabbit secondary antibody
1
Nanobody-based Miro1 Visualization
2
Imaging Miro1 localization in HeLa cells
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HeLa cells were seeded at 1 x 10 4 per well in μClear 96 well plates (Greiner). Next day, the cells were transfected with plasmids coding for GFP-Miro1 or a GFP-tagged non-related protein. 24 h post transfection, the cells were washed twice with PBS and fixed with 4% (w/v) paraformaldehyde (PFA) in PBS for 10 min at RT and blocked with 5% BSA in TBST for 30 min at RT. Incubation with purified Miro1 Nbs or AF647 conjugated Nbs (100 -200 nM in 5% BSA in TBST) or rabbit anti-Miro1 antibody (# NBP1-89011, Novus Biologicals) was performed overnight at 4 °C. Unbound nanobodies were removed by three washing steps with TBST. Unlabelled Miro1 Nbs were detected by addition of a Cy5-conjugated Goat Anti-Alpaca IgG (Jackson Immunoresearch) according to manufacturer's guidelines. For Miro1 antibody detection, an AF647 conjugated goat anti-rabbit secondary antibody (Invitrogen) was used.
Nuclei were subsequently stained with 4', 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and images were acquired immediately afterwards with an ImageXpress™ Micro Confocal High Content Screening system (Molecular Devices) at 40x magnification.
Nuclei were subsequently stained with 4', 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and images were acquired immediately afterwards with an ImageXpress™ Micro Confocal High Content Screening system (Molecular Devices) at 40x magnification.
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