Evaluating the Intracellular Distribution of Virus-Derived Peptide-Modified Antibody Conjugates

Abstract

Source: Beaudoin, S. et al., Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting. J. Vis. Exp. (2018)

This video demonstrates the process of assessing the intracellular distribution of virus-derived peptide-modified antibody conjugates using confocal microscopy. The viral-derived nuclear localization sequence directs antibody-conjugates within the cytoplasm, confirmed through immunostaining and fluorescence measurement.

Protocol

1. Antibody Peptide Conjugation NOTE: Cholic acid linked to NLS (ChAcNLS) can be synthesized at any commercial peptide manufacturer or university-affiliated peptide synthesis service platform. In a 1.7 mL microcentrifuge tube, prepare a 10 mg/mL (~1 mg total antibody) solution of 7G3 in phosphate buffered saline (PBS), pH 7.6. Dissolve sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) in dimethyl sulfoxide…

Representative Results

Figure 1: Schematic of AC Cell Treatment Approach and Subsequent Processing for Analysis by Confocal Microscopy and for Analysis of the Cell Fractionation Quality. Red arrows correspond to essential steps 2.2 and 2.3. 

Materials

Sulfo-SMCC	Thermo Scientific	22122	There are many homo- and hetero-bifunctional maleimide crosslinkers to choose from.
Amicon Ultra-0.5 mL Centrifugal Filters	EMD Millipore	UFC505096	There are pack sizes of 8, 24, and 96. Choose according to your needs.
Precision Plus Protein Kaleidoscope Standards	BioRad	1610375EDU	Mulicolor recombinant proteins from 10-250 kDa.
Trypsin-EDTA (0.25%), phenol red	Thermo Scientific	25200056	100 or 500 mL volumes to choose from.
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate	Thermo Scientific	A-21235	1-10 μg/mL recommended
NuPAGE LDS Sample Buffer (4x)	Thermo Scientific	NP0007
2-Mercaptoethanol	Sigma Aldrich	M3148-25ML
TF-1a cells	ATCC	ATCC CRL-2003
RPMI 1640 medium	ATCC	ATCC 30-2001
FluoView FV1000 Confocal Microscope	Olympus
Fluoview Software	Olympus		www.olympus-lifescience.com