Journal: Bio-protocol

Article Title: New Approach to Detect and Isolate Rhamnogalacturonan-II in Arabidopsis thaliana Seed Mucilage

doi: 10.21769/BioProtoc.5433

Figure Lengend Snippet: (A) Using the protocol described by Chormova et al. [6], the isolated RG-II can be visualised by electrophoresis. The figure presents RG-II isolated from Arabidopsis Col-0 and three different allelic mutants of the AtBXL1 gene. As molecular markers, dimeric RG-II (dRG-II) was included, obtained from Arabidopsis leaves through digestion with endo-polygalacturonase. The monomeric form of RG-II (mRG-II) was generated by acid treatment of the dimer. (B) The figure shows a re-dimerisation assay used to confirm that the isolated domain corresponds to RG-II. To achieve this, the dimeric form was first monomerised via acidic treatment, then subjected to re-dimerisation, as described by [6]. Lanes: 1) 1.6 μg of dRG-II; 2) 1.6 μg of mRG-II (dRG-II treated with HCl); 3) 1.6 μg of mRG-II; 4) 1.6 μg of mRG-II + Pb 2+ + H 3 BO 3 ; 5) 1.6 μg of mRG-II + Pb 2+ ; 6) 1.6 μg of mRG-II + H 3 BO 3 ; 7) 2.4 μg each of dimeric and monomeric RG-II prepared from Arabidopsis leaves (used as markers). Reactions 3–6 were prepared in a final volume of 10 µL using 50 mM sodium acetate buffer (pH 4.8), 0.5 mM Pb(NO 3 ) 2 , and 1.2 mM H 3 BO 3 . All samples were incubated overnight at room temperature.

Article Snippet: Electrophoresis system (Bio-Rad, model: Mini-PROTEAN ® Tetra Cell, catalog number: 165-8004) 4.

Techniques: Isolation, Electrophoresis, Generated, Incubation