eoe sub articles

EoE Sub Articles

1. Nuclear extraction and cell fixation
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Prepare 4% (v/v) paraformaldehyde (PFA): dissolve 10 mL of 16% PFA aqueous solution in 30 mL PBS. Mi...

immunofluorescence-based visualization of dna repair protein interactions

Source: de la Pe&#241;a Avalos, B., et al., <a href="https://app.jove.com/v/61447">Visualization of DNA Repair Proteins Interaction by Immunofluorescence.</a> J. Vis. Exp. (2020).
In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins &#947;H2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

<img alt="Figure 1" src="/files/ftp_upload/21438/21438_Figure1.jpg" /> 
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 &#956;m and (B) 40x magnification; scale bar = 10 &#956;m.

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Source: de la Pe&#241;a Avalos, B., et al., Visualization of DNA Repair Proteins Interaction by Immunofluorescence. J. Vis. Exp. (2020).
In this video, we ...

Radiation-induced DNA double-strand breaks, or DSBs, initiate DNA repair protein recruitment at the damaged site &#8212; activating DNA repair.
To detect the DNA repair proteins &#947;H2AX and 53BP1 at DSB sites using indirect immunofluorescence, take coverslips containing epithelial cells. Irradiate a subset of the cells. Incubate, allowing the control non-irradiated and irradiated cells to grow for specific times.
Treat with a buffer to extract the cell nuclei. Fix the cell nuclei to preserve their morphology. Treat with a blocking solution to minimize non-specific primary antibody binding.
Add primary antibodies targeting the two DNA repair proteins. The antibodies bind to the respective proteins.
Incubate with secondary antibodies tagged with red and green fluorophores, respectively. The secondary antibodies &#8212; specific for each primary antibody &#8212; bind to their targets. Add mounting medium containing DAPI on a glass slide. Mount the coverslips on it for imaging.
DAPI &#8212; a DNA-binding dye &#8212; binds to the AT-rich minor grooves of the double-stranded DNA, counterstaining the nuclei. Visualize the cell nuclei using a fluorescence microscope.
Compared to non-irradiated cell nuclei, irradiated nuclei exhibit multiple &#947;H2AX foci that decrease over time, indicating progressive DNA repair in cells grown for longer periods.
Overlapping red and green fluorescence signals appear yellow, indicating the co-localization of &#947;H2AX and 53BP1 at the DSB site following irradiation.

immunofluorescence-based-visualization-dna-repair-protein

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins &#947;H2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21438/21438_Figure1.jpg" /> 
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 &#956;m and (B) 40x magnification; scale bar = 10 &#956;m.

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

In This Article

Overview

Protocol

Representative Results