Alexa fluor 430 nhs ester

All chemicals
and reagents used were of analytical grade. Poly-l-lysine
(PLL) hydrochloride (M.W. >12 000, cutoff by dialysis) was
purchased from PEPTIDE INSTITUTE. PEGylated, long-chain SPDP cross-linker
(PEG4-SPDP) and Alexa Fluor 430 NHS ester were obtained from Thermo
Fisher Scientific. Citrate-coated colloidal silver nanoparticles (AgNPs)
with a mean diameter of 80 nm were purchased from NanoComposix. Amino-functionalized
polystyrene (PS) nanospheres with a mean diameter of 100 nm were obtained
from Polysciences. Bissulfosuccinimidyl suberate (BS3) was supplied
by Dojindo. Dimethyl sulfoxide (DMSO) was purchased from Fujifilm
Wako Pure Chemical Corp. Tris(2,2′-bipyridyl)ruthenium(II)
chloride hexahydrate, cysteamine, and (3-aminopropyl)triethoxysilane
(APTES) were purchased from Tokyo Chemical Industry Co., Ltd (TCI).

The monoclonal anti-HA antibody was labeled with the reactive fluorescent probe Alexa Fluor 430 NHS Ester (Succinimidyl Ester) purchased from Thermo Fisher Scientific. In brief, the antibody was concentrated and dialyzed in reaction buffer (0.1 M sodium bicarbonate buffer, pH 8.3). 0.2 mg of antibody was mixed with Alexa Fluor 430 with a molar ratio of 12:1 (dye:antibody) in a final volume of 0.125 ml and incubated at RT for 1 h. To remove unreacted fluorescence probe and change buffer, the anti-HA–Alexa Fluor 430 complex was purified using a Sephadex G25 column (GE Healthcare) preequilibrated with 20 mM sodium phosphate, pH 7.4, and 0.1 M NaCl. 50-µl fractions were collected; the absorption spectra between 220 and 600 nm were measured; and the degree of labeling was 4, calculated according to the manufacturer’s protocol.

Tubulin‐AF was prepared using Alexa Fluor™ 430 NHS ester (Thermo Fisher Scientific) according to the standard procedure.51 The labeling ratio of tubulin‐AF was determined by measuring the absorbance of the protein and Alexa Fluor 430 at 280, and 430 nm, respectively.