Scotch tape (single sided Scotch ® Magic TMTape 810) NCS18 silicon probe (75 kHz, 3.5 N/m) (Mikromasch, catalog number: HQ:NSC18/Cr-Au-15) Plastic petri dishes (100 x 15 mm dimension) (VWR International, catalog number: 25384302 ) Kimwipes (VWR International, catalog number: 470173498 ) Highly ordered pyrolytic graphite (square shape 5 mm (L) x 5 mm (W) x 1 mm (H) in dimension) (Structure Probe, catalog number: 476HPAB ) Sodium acetate (NaOAc, anhydrous, purity ≥ 99.0%) (Sigma-Aldrich, catalog number: S8750 )Īcetic acid (HOAc, ACS reagent, purity ≥ 99.7%) (Sigma-Aldrich, catalog number: 320099 )ħ5% ethanol (prepared from 100% ethanol, ACS reagent, purity ≥ 99.5%) (Sigma-Aldrich, catalog number: 459844 ) (2011b).īovine serum albumin (BSA, lyophilized powder, crystallized, purity ≥ 98.0%) (Sigma-Aldrich, catalog number: 05470 ) For a detailed extensin extraction protocol see Lamport et al.
Monomeric extensin proteins extracted from different plant cell suspension culture lines. Keywords: Plant cell wall, Structural glycoprotein, Extensin, Self-assembly, Atomic force microscope The protocol elaborated here provides an approach to studying the self-assembly of extensins and potentially of other cell wall components in vitro using AFM. A detailed protocol for extraction of extensin and other wall structural proteins has been described earlier (Lamport et al., 2011b). Extensins are recalcitrant to purification as they are rapidly cross-linked into a covalent network after entering the cell wall but there exists a short time window in which newly synthesized extensin monomers can be extracted (Smith et al., 1984 Smith et al., 1986) by salt elution. As essential members of the HRGP superfamily, extensins (EXTs) presumably function in the cell wall by assembling into positively charged protein scaffolds (Cannon et al., 2008) that direct the proper deposition of other wall polysaccharides, especially pectins, to ensure correct cell wall assembly (Hall and Cannon, 2002 Lamport et al., 2011a).
Hydroxyproline-rich glycoproteins (HRGPs) are major protein components in dicot primary cell walls and generally account for more than 10% of the wall dry weight.
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