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* These authors contributed equally
This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.
Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.
Our genome is constantly being challenged by various DNA damaging agents. These assaults can derive from environmental sources, such as UV light or irradiation, as well as from endogenous sources, such as metabolic by-products caused by oxidative stress or replication errors1,2. These lesions can affect the integrity of either one or both DNA strands, and if the generated errors become persistent, it frequently leads to translocations and genome instability, which may result in tumorigenesis3,4. To maintain genome integrity, multiple repair systems hav....
1. Immunodetection of specific proteins
Studying site-directed DSB-induced repair processes in cells can be achieved via either stable or transient transfection. However, it should be noted that stable transfection ensures a homogenous cell population, which gives a unified and thus more reliable cellular response. In the case of transient transfection, only a small proportion of the cell population takes up and maintains the plasmid, which introduces diversity into the experiment. Establishing ER-I-PpoI or ER-AsiSI endonuclease-based cell systems require.......
Although DNA repair is a relatively recent research field, our knowledge is rapidly expanding with the help of various biochemical and microscopic methods. Preserving genetic information is crucial for cells since mutations occurring in genes involved in repair processes are among the leading causes of tumorigenesis and therefore elucidating the key steps of DNA repair pathways is essential.
Biochemical techniques (i.e., western blot, immunoprecipitation, mass-spectrometry, etc.) require large.......
This research was funded by the National Research, Development and Innovation Office grant GINOP-2.3.2-15-2016-00020, GINOP-2.3.2-15-2016-00036, GINOP-2.2.1-15-2017-00052, EFOP 3.6.3-VEKOP-16-2017-00009, NKFI-FK 132080, the János Bolyai Research Scholarship of the Hungarian Academy of Sciences BO/27/20, ÚNKP-20-5-SZTE-265, EMBO short-term fellowship 8513, and the Tempus Foundation.
....Name | Company | Catalog Number | Comments |
4-OHT | Sigma Aldrich | H7904 | |
Agarose | Lonza | 50004 | |
Antibiotic-Antimycotic Solution (100×), Stabilized | Sigma Aldrich | A5955 | |
Anti-gamma H2A.X (phospho S139) antibody | Abcam | ab26350 | |
Bovine Serum Fraction V albumin | Biosera | PM-T1725 | |
TrackItâ„¢ Cyan/Yellow Loading Buffer | Thermo Fisher Scientific | 10482035 | |
DMEM with 1.0 g/L Glucose, without L-Glutamine | Lonza | 12-707F | |
Doxycycline | Sigma Aldrich | D9891 | |
Dynabeadsâ„¢ M-280 Sheep Anti-Mouse IgG | Invitrogen | 11202D | |
Dynabeadsâ„¢ M-280 Sheep Anti-Rabbit IgG | Invitrogen | 11204D | |
EDTA | Sigma Aldrich | E6758 | |
EGTA | Sigma Aldrich | E3889 | |
Ethanol | Molar Chemicals | 02910-101-340 | |
Fetal Bovine Serum (South America Origin), EU-approved | Gibco | ECS0180L | |
Formaldehyde 37% solution free from acid | Sigma Aldrich | 1.03999 | |
GlutaMAXâ„¢ Supplement | Thermo Fisher Scientific | 35050038 | |
Glycine | Sigma Aldrich | 50046 | |
IPure kit v2 | Diagenode | C03010015 | |
Isoamyl alcohol | Sigma Aldrich | W205702 | |
LiCl | Sigma Aldrich | L9650 | |
NaCl | Sigma Aldrich | S5886 | |
Na-DOC | Sigma Aldrich | D6750 | |
NaHCO3 | Sigma Aldrich | S5761 | |
Neocarzinostatin from Streptomyces carzinostaticus | Sigma Aldrich | N9162 | |
NP-40 | Sigma Aldrich | I8896 | |
PBS Powder without Ca2+, Mg2+ | Sigma Aldrich | L182-50-BC | |
Phenol | Sigma Aldrich | P4557 | |
PIPES | Sigma Aldrich | P1851 | |
Polysorbate 20 (Tween 20) | Molar Chemicals | 09400-203-190 | |
KCl | Sigma Aldrich | P5405 | |
ProLongâ„¢ Gold Antifade Mountant with DAPI | Thermo Fisher Scientific | P36935 | |
Protease Inhibitor Cocktail Set I | Roche | 11873580001 | |
Proteinase K | Sigma Aldrich | P2308 | |
P-S2056 DNAPKcs antibody | Abcam | ab18192 | |
RNase A | Roche | 10109169001 | |
CH3COONa | Sigma Aldrich | S2889 | |
SDS | Sigma Aldrich | L3771 | |
Tris Acetate-EDTA buffer | Sigma Aldrich | T6025 | |
Tris-HCl | Sigma Aldrich | 91228 | |
TRITON X-100 | Molar Chemicals | 09370-006-340 | |
Trypsin from porcine pancreas | Sigma Aldrich | T4799 | |
Trypsin-EDTA (0.5%), no phenol red | Gibco | 15400054 |
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