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.
Divulgazioni
The authors have nothing to disclose.
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.
Evaluating the Intracellular Distribution of Virus-Derived Peptide-Modified Antibody Conjugates. J. Vis. Exp. (Pending Publication), e22126, doi: (2024).