Halo-ligands conjugated to Alexa Fluor 488, Janelia Fluor 549, and Janelia Fluor 646 fluorophores (Promega) were reconstituted to 1 mM in DMSO. Halo-tagged proteins were mixed with fluorophores in a 1:1 concentration ratio, then incubated on ice for 1–2 hours in the dark. After Halo-ligand conjugation reactions were complete, unreacted fluorophores were removed with micro-spin columns loaded with dye removal purification resin (Pierce) at 4 °C. Proteins were then diluted to 2–8 μM in MBOS (Motility Buffer + Oxygen Scavengers: 20 mM MOPS pH 7.4, 5 mM MgCl2, 0.1 mM EGTA, 50 mM KCl, 15 mM glucose, 50 mM DTT, 20 μg/mL catalase (Sigma C3515), and 100 μg/mL glucose oxidase (Sigma G6125)) + 1–4 mM ATP. Aggregates were removed by ultracentrifugation at 4 °C at 100,111 × g in a TLA100 rotor (Beckman). Fresh Halo-fluorophore conjugated proteins were prepared for each experiment.
Alexa fluor 488
Manufactured by Promega
Alexa Fluor 488 is a fluorescent dye manufactured by Promega. It is a small molecule that can be used to label proteins, nucleic acids, and other biomolecules. Alexa Fluor 488 has an excitation maximum at 490 nm and an emission maximum at 525 nm, making it suitable for use in a variety of fluorescence-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Glucose oxidase, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Janelia fluor 549, by Promega (1 mentions)
Janelia fluor 646, by Promega (1 mentions)
Tla100 rotor, by Beckman Coulter (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Elyra7 super resolution structured illumination microscope, by Zeiss (1 mentions)
3 protocols using alexa fluor 488
1
Halo-Ligand Fluorophore Conjugation Protocol
2
PEG Biomolecule Passivation Efficacy
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Each of the 97 PEG biomolecules that we tested
(Table S1 ) was applied to the cleaned and
dried surfaces as a 1 μL droplet, and the surfaces were incubated
for 1 h at room temperature in a humidified chamber. Then, the PEG
was removed by aspiration, and the surfaces were washed once with
0.1 M phosphate-buffered saline (PBS), pH 7.4. Finally, the HaloTag-fused
protein was added, and the surfaces were incubated for 1 h at room
temperature. The surfaces were incubated with primary antibody against
the HaloTag (anti-HaloTag polyclonal antibody, cat. G9281, Promega)
and subsequently with a secondary antibody conjugated to a fluorophore
(Alexa Fluor 488, cat. A-11008, Invitrogen). To quantify the effectiveness
of a variety of PEG solutions, we measured the immunofluorescence
intensity of noncoated surfaces and PEG-coated areas of the surfaces
and calculated the efficiency of passivation as a percentage.
(
dried surfaces as a 1 μL droplet, and the surfaces were incubated
for 1 h at room temperature in a humidified chamber. Then, the PEG
was removed by aspiration, and the surfaces were washed once with
0.1 M phosphate-buffered saline (PBS), pH 7.4. Finally, the HaloTag-fused
protein was added, and the surfaces were incubated for 1 h at room
temperature. The surfaces were incubated with primary antibody against
the HaloTag (anti-HaloTag polyclonal antibody, cat. G9281, Promega)
and subsequently with a secondary antibody conjugated to a fluorophore
(Alexa Fluor 488, cat. A-11008, Invitrogen). To quantify the effectiveness
of a variety of PEG solutions, we measured the immunofluorescence
intensity of noncoated surfaces and PEG-coated areas of the surfaces
and calculated the efficiency of passivation as a percentage.
3
Visualizing Protein Patterns by Immunofluorescence
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Proteins immobilized to patterns were detected by immunofluorescence
microscopy using a polyclonal primary antibody against the HaloTag
epitope of HT-EphA2 (anti-HaloTag, cat. G9281, Promega) and counterstaining
with anti-rabbit secondary antibody conjugated to Alexa Fluor 488
(cat. A-11008, Invitrogen). Each antibody incubation was performed
for 1 h at room temperature, and then, the surfaces were washed three
times in 1× PBS. The samples were mounted using ProLong Gold
antifade mountant (cat. P10144, Thermo Fisher Scientific). The samples
were analyzed with an LSM880 laser scanning confocal microscope (Carl
Zeiss AG) with a super-resolution Airyscan detector and with an Elyra
7 super-resolution structured illumination microscope (Carl Zeiss
AG).
microscopy using a polyclonal primary antibody against the HaloTag
epitope of HT-EphA2 (anti-HaloTag, cat. G9281, Promega) and counterstaining
with anti-rabbit secondary antibody conjugated to Alexa Fluor 488
(cat. A-11008, Invitrogen). Each antibody incubation was performed
for 1 h at room temperature, and then, the surfaces were washed three
times in 1× PBS. The samples were mounted using ProLong Gold
antifade mountant (cat. P10144, Thermo Fisher Scientific). The samples
were analyzed with an LSM880 laser scanning confocal microscope (Carl
Zeiss AG) with a super-resolution Airyscan detector and with an Elyra
7 super-resolution structured illumination microscope (Carl Zeiss
AG).
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