Evaluating the Intracellular Distribution of Virus-Derived Peptide-Modified Antibody Conjugates
Published: April 30, 2024
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.
Açıklamalar
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. |
| 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 |
Etiketler
Bu Makaleden Alıntı Yapın
Evaluating the Intracellular Distribution of Virus-Derived Peptide-Modified Antibody Conjugates. J. Vis. Exp. (Pending Publication), e22126, doi: (2024).