Immunofluorescence-Based Visualization of DNA Repair Protein Interactions
Published: June 29, 2023
Abstract
Source: de la Peña Avalos, B., et al., Visualization of DNA Repair Proteins Interaction by Immunofluorescence. J. Vis. Exp. (2020).
In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins γH2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.
Protocol
1. Nuclear extraction and cell fixation Prepare stock solutions: 0.2 M PIPES (pH 6.8), 5 M NaCl, 2 M sucrose, 1 M MgCl2, 0.1 M EGTA (pH 8.0). Prepare nuclear extraction buffer (NEB): dissolve 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0) and 0.5% (v/v) Triton X-100 in ddH2O. Mix until dissolved completely. Prepare 4% (v/v) paraformaldehyde (PFA): dissolve 10 mL of 16% PFA aqueous solution in 30 mL PBS. Mi…
Representative Results
Figure 1. Nuclear extraction. Representative images of cells prior to (left) and post (right) nuclear extraction. Cytoplasm should be digested but the nuclear structure should remain intact post-extraction (right). (A) 20x magnification; scale bar = 20 μm and (B) 40x magnification; scale bar = 10 μm.
Divulgaciones
The authors have nothing to disclose.
Materials
| Coverglass #1, 18 mm round (coverslips) | Neuvitro | NC0308920 | Coverslips need to be cleaned and sterilized prior using, with HCl or ethanol. |
| DPBS, no calcium, no magnesium | Gibco | 14190144 | PBS for tissue culture |
| SlowFade Diamond Antifade Mountant with DAPI | Invitrogen | S36973 | 300 nM DAPI with VECTASHIELD Antifade Mounting Medium can be used instead. |
| Bovine serum albumin (BSA) | Sigma-Aldrich | A3059 | Heat-shock fraction is recommended, to avoid precipitation/background. |
| Triton X-100 | AmericanBio | AB02025 | Nuclear extraction buffer. |
| Superfrost Plus Microscope Slides | Fisherbrand | 1255015 | Polysine Slides can be used instead. |
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Immunofluorescence-Based Visualization of DNA Repair Protein Interactions. J. Vis. Exp. (Pending Publication), e21438, doi: (2023).