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  • An efficient immunodetection method for histone modifications in plants.

An efficient immunodetection method for histone modifications in plants.

Plant methods (2013-12-18)
Geovanny Nic-Can, Sara Hernández-Castellano, Angela Kú-González, Víctor M Loyola-Vargas, Clelia De-la-Peña
ABSTRACT

Epigenetic mechanisms can be highly dynamic, but the cross-talk among them and with the genome is still poorly understood. Many of these mechanisms work at different places in the cell and at different times of organism development. Covalent histone modifications are one of the most complex and studied epigenetic mechanisms involved in cellular reprogramming and development in plants. Therefore, the knowledge of the spatial distribution of histone methylation in different tissues is important to understand their behavior on specific cells. Based on the importance of epigenetic marks for biology, we present a simplified, inexpensive and efficient protocol for in situ immunolocalization on different tissues such as flowers, buds, callus, somatic embryo and meristematic tissue from several plants of agronomical and biological importance. Here, we fully describe all the steps to perform the localization of histone modifications. Using this method, we were able to visualize the distribution of H3K4me3 and H3K9me2 without loss of histological integrity of tissues from several plants, including Agave tequilana, Capsicum chinense, Coffea canephora and Cedrela odorata, as well as Arabidopsis thaliana. There are many protocols to study chromatin modifications; however, most of them are expensive, difficult and require sophisticated equipment. Here, we provide an efficient protocol for in situ localization of histone methylation that dispenses with the use of expensive and sensitive enzymes. The present method can be used to investigate the cellular distribution and localization of a wide array of proteins, which could help to clarify the biological role that they play at specific times and places in different tissues of various plant species.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Sodium citrate dihydrate, ≥99%, FG
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Anti-Histone H3 Antibody, CT, pan, serum, Upstate®
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Anti-trimethyl-Histone H3 (Lys4) Antibody, clone 15-10C-E4, rabbit monoclonal, clone 15-10C-E4, Upstate®, from rabbit
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Acetic acid, glacial, ACS reagent, ≥99.7%
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Xylenes, histological grade
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Anti-dimethyl-Histone H3 (Lys9) Antibody, Upstate®, from rabbit
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Sodium phosphate dibasic, for molecular biology, ≥98.5% (titration)