Full-length budding yeast Cin8 with a C-terminal monomeric green fluorescent protein (GFP) tag (Cin8-mGFP) was prepared as described (12 (link)). Full-length X. laevis Eg5 with a C-terminal GFP tag was prepared as described (9 (link)), but imidazole was omitted from the final buffer. Full-length, non-fluorescently tagged Drosophila melanogaster kinesin-1 was prepared as described (30 (link), 31 (link)), with the exception that the elution buffer was not added as a gradient. For motor proteins, we state monomer concentrations throughout, unless indicated otherwise. Tubulin was purified from porcine brain, as described in (32 (link)). To generate biotinylated tubulin, Alexa647-tubulin, or NEM-tubulin, tubulin was covalently labeled with either EZ-Link NHS-LC-LC-biotin (21343, Life Technologies, Carlsbad, CA), Alexa Fluor 647-NHS (A-20006, Life Technologies), or N-(ethylmaleimide) (NEM) (Sigma-Aldrich, St. Louis, MO), respectively, essentially as described previously (33 (link)). All proteins were aliquoted in small aliquots, snap frozen, and stored in liquid nitrogen.
Alexa fluor 647 nhs
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Alexa Fluor 647-NHS is a fluorescent dye used for labeling proteins and other biomolecules. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, making it suitable for detection in the red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
N ethylmaleimide nem, by Merck Group (1 mentions)
C reactive protein (crp), by Merck Group (1 mentions)
N succinimidyl 3 2 pyridyldithio propionate, by Merck Group (1 mentions)
Alexa fluor 647 c2 maleimide, by Thermo Fisher Scientific (1 mentions)
D luciferin, by GoldBio (1 mentions)
Slide a lyzer g2, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Nhs fitc, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Expi293, by Thermo Fisher Scientific (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
100 kda mwco spin concentrator, by Merck Group (1 mentions)
Safeblue staining, by Thermo Fisher Scientific (1 mentions)
V 650, by Jasco (1 mentions)
Prism 7, by GraphPad (1 mentions)
Agilent prep c18 column, by Agilent Technologies (1 mentions)
Agilent 1200 system, by Agilent Technologies (1 mentions)
Zeba desalting column, by Thermo Fisher Scientific (1 mentions)
19 protocols using alexa fluor 647 nhs
1
Labeling and Purification of Motor Proteins
2
Fluorescent Labeling of GSAO and CRP
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GSAO and control compound 4‐(N‐(S‐glutathionylacetyl)amino)benzoic acid were synthesized previously as described25 and conjugated to amine‐reactive succinimidyl ester Alexa Fluor 647‐NHS (Life Technologies). Confirmation of GSAO activity and batch to batch validation testing after conjugation were performed as previously described.17 , 26 Monomeric collagen‐related peptide (CRP, Auspep) was cross‐linked using N‐succinimidyl 3‐(2‐pyridyldithio)propionate (Sigma‐Aldrich) to produce CRP‐xL. Reagents are available on request.
3
Fluorescent Conjugation Protocol
4
Antibody-Fluorophore Conjugation Protocol
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Example 2
Antibody-TCO conjugates were further modified with fluorescent dye. Antibody at ˜0.4 μg/mL was combined with 1 M sodium bicarbonate to obtain a final buffer concentration of 100 mM sodium bicarbonate in PBS. To this solution, 1.2 μL of 3 mM Alexa Fluor 647-NHS (Thermo) was added and the resulting mixture was incubated at room temperature for 1 hour, protected from ambient light. Excess fluorophore was removed via filtration in a centrifugal filter, washing repeatedly with PBS. This procedure typically resulted in 4-7 fluorophore molecules per full length IgG antibody.
5
Fluorescent Labeling of HaloTag Ligand
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HaloTag-Ligand-Amine (Promega, #P6741) was reconstituted in N,N-dimethylformamid (DMF; Sigma-Aldrich, #70547) to a concentration of 10 g/l. 250 μg HaloTag-Ligand Amine was conjugated to Alexa Fluor 647-NHS (Thermo Fisher, #A20006) by adding 150 μg of NHS-dye from a 10 g/l stock solution in DMF. The total reaction volume was adjusted to 120 μl with DMF, then DIPEA Base (Sigma-Aldrich, #387649) was added at 1:100. The reaction was incubated under gentle agitation and protected from light at room temperature for 3 hr. The reaction mixture was purified by reverse-phase high-performance liquid chromatography (Jasco) using a biphenyl column (Kinetex, #00F-4622-E0) with a solvent gradient of 0–60% (vol/vol) acetonitrile (Sigma-Aldrich, #34851) in 0.1% trifluoroacetic acid (TFA; Sigma-Aldrich, #T6508) in water run over 30 min. Collected product fractions were dried in a speed-vac (Thermo Fisher, #SPD111V) and dissolved in DMSO.
6
Antibody Fluorescent Labeling Protocol
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Example 2
Antibody-TCO conjugates were further modified with fluorescent dye. Antibody at ˜0.4 μg/mL was combined with 1 M sodium bicarbonate to obtain a final buffer concentration of 100 mM sodium bicarbonate in PBS. To this solution, 1.2 μL of 3 mM Alexa Fluor 647-NHS (Thermo) was added and the resulting mixture was incubated at room temperature for 1 hour, protected from ambient light. Excess fluorophore was removed via filtration in a centrifugal filter, washing repeatedly with PBS. This procedure typically resulted in 4-7 fluorophore molecules per full length IgG antibody.
7
Alexa Fluor 647 Conjugation to Magnetic Beads
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Alexa Fluor 647 was immobilized onto Dynabeads M-270 amine magnetic beads (Thermo Fisher Scientific) using standard amine reactive chemistry. 400 μL of amine beads were washed three times with 400 μL 0.1% Tween-20 in PBS. 1 mg Alexa Fluor 647 NHS ester (Thermo Fisher Scientific) was resuspended in dimethylformamide (DMF) to a final concentration of 10 μg/μL. The washed beads were resuspended in a 400 μL solution comprising 24 μL Alexa Fluor 647 NHS-ester stock in 1X PBS. The bead mixture was incubated for 2 hrs at room temperature (RT) with rotation. Beads were then washed three times and resuspended with 400 μL 1X SELEX buffer.
8
Fluorescent Labeling of MCF10A Cells
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Flash frozen MCF10A cell pellets were washed with protease inhibitor cocktail supplemented PBS and sonicated on ice until homogenous. 20 μg of protein from the homogenized samples were then labeled with Alexa Fluor 555-NHS 86 (link). A reference sample was prepared by pooling equal amounts (by total protein) of all samples and labeling with Alexa Fluor 647-NHS (Thermo Fisher). Table S1 summarizes the print list for the experiment Printing, hybridization, and data analysis 87 (link).
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9
Cellular Internalization of Engineered Extracellular Vesicles
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To better evaluate the cellular internalization, mEVs were loaded with 0.5 mM AO by SEAL, and labelled with Alexa fluor 647‐NHS (Thermo Scientific, USA) via the NHS‐amine reaction. As for the preparation of ligand conjugated mEVs, facile reduction of surface disulfides on mEVs was conducted followed by the click reaction with maleimide modified transferrin (Sigma‐Aldrich) (Chen et al., 2021 (link)). Then AO‐mEVs‐AF647, AO‐mEVs‐AF647‐Tf and the magnetic purified AO‐mEVs‐AF647‐Tf were incubated with HepG2 cells for 9 h, respectively. Once removing the uninternalized mEVs and washing with PBS, the cells were stained with Hoechst 33342 and imaged on a Leica SP8 confocal microscope. Regarding FCM analysis, three mEV formulations, as well as the blank mEVs, were incubated with HepG2 cells for 12 h. After removing the uninternalized mEVs, the fluorescence intensity of the cells was measured immediately using a flow cytometer (FACS Aria, BD).
10
Recombinant Antibody Generation and Purification
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Recombinant antibodies were generated using the Expi293 (ThermoFisher) or Expi293 FUT8−/− system using previously described protocols (47 (link)). Briefly, an equal ratio of heavy- and light-chain plasmids was complexed with ExpiFectamine in OptiMEM and added to Expi293 cells in culture at 3 × 106 cells/ml. Enhancer 1 and Enhancer 2 were added 20 h after transfection. After 6 d, recombinant IgG antibodies were purified from cell-free supernatants by affinity purification using protein G sepharose beads (GE Healthcare), dialyzed in PBS, filter-sterilized (0.22 μm), concentrated with 100 kDa MWCO spin concentrator (Millipore), purified with Superdex 200 Increase 10/300 GL (GE Healthcare), and finally assessed by SDS–PAGE followed by SafeBlue staining (ThermoFisher). All antibody preparations were more than 95% pure and endotoxin levels were less than 0.05 EU mg−1, as measured by the Limulus amebocyte lysate assay. Purified IgG was fluorescently labeled with Alexa Fluor 647-NHS or FITC-NHS (ThermoFisher) at a 15-fold molar excess for 1 h at room temperature and double-dialyzed into PBS.
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