In vitro analysis of CBM and antibody binding was carried out using ELISAs as described (12 (link),17 (link)). For antibody/CBM capture ELISAs, 10-fold serial dilutions of 100 μg/ml tamarind xyloglucan (Megazyme) in PBS were coated (100 μl) on to microtitre plates (Nunc, Denmark). LM15 was used at 1:20 dilution of hybridoma cell culture supernatant in MP/PBS. CtCBM3a was used at 10 μg/ml in MP/PBS. Anti-rat-IgG-peroxidase (A9037 Sigma-Aldrich) and anti-his-peroxidase (A7058 Sigma-Aldrich) were used as secondary antibodies for LM15 and CtCBM3a respectively. For competitive-inhibition assessment of recognition of polysaccharides/oligosaccharides microtitre plates were coated with 0.5 μg/ml xyloglucan to act as immobilised antigen. After blocking with MP/PBS and washing, six ten-fold serial dilutions from 50 μg/ml of xyloglucan from tamarind seeds (P-XYGLN Megazyme), xyloglucan heptasaccharide (O-X3G4 Megazyme), guar galactomannan medium viscosity (P-GGMMV Megazyme) and cellohexaose (O-CHE Megazyme) haptens were prepared and 50 μl added to microtritre plate wells. Immediately after, 50 μl of 1:50 dilution of LM15 antibody or CBMs at 20 μg/ml were added to equivalent sets of microtitre plate wells containing the soluble xyloglucan or oligosaccharide haptens and incubated for 1.5 h. Probe binding was determined as described above.
P xygln
Manufactured by Megazyme
Sourced in Ireland
P-XYGLN is a laboratory testing kit designed for the quantitative determination of xyloglucan in plant materials. It is a sensitive and specific assay that utilizes an enzymatic reaction to measure the xyloglucan content.
Automatically generated - may contain errors
Lab products found in correlation
Tamarind xyloglucan, by Megazyme (1 mentions)
A9037, by Merck Group (1 mentions)
Microtitre plates, by Thermo Fisher Scientific (1 mentions)
A7058, by Merck Group (1 mentions)
P waxyl, by Megazyme (1 mentions)
P xylnbe, by Megazyme (1 mentions)
P maniv, by Megazyme (1 mentions)
Whatman glass microfiber filter, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
3 protocols using p xygln
1
In vitro Analysis of CBM and Antibody Binding
2
Enzymatic Hydrolysis of Plant Polysaccharides
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Beechwood xylan (P-XYLNBE, Megazyme, Bray, Ireland), wheat flour arabinoxylan (P-WAXYL, Megazyme, Bray, Ireland), tamarind xyloglucan (P-XYGLN, Megazyme, Bray, Ireland), and ivory nut mannan (P-MANIV, Megazyme, Bray, Ireland) were used as substrates to test secretome enzymatic activities. Substrates were mixed with the secretomes at the final concentration of 1% (w/v). Samples were incubated under a constant shaking rate (800 rpm) using an LLG-uniTHERMIX2 pro thermo shaker (LLG. Meckenheim Germany). The digestion temperature and pH were set at the optimal value for each secretome. Hydrolysis was stopped after 1, 2, 3, 4, 6, and 24 h by boiling the reaction at 100 °C for 5 min to inactivate the enzymes.
3
RNA-seq of N. crassa metabolism
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Media shift experiments were performed in triplicate and followed the procedure described earlier in Coradetti et al. [16 (link)] and Znameroski et al. [18 (link)] to ensure optimal comparability with the previously published RNA-seq datasets. First, using shake flasks (200 rpm), N. crassa cultures were pre-grown from conidia for 16 h in 100 mL of VMM [100 ] with 2% sucrose. Next, the mycelia were passed over a Whatman glass microfiber filter and washed three times with VMM without a carbon source (NoC). The mycelial mass was then transferred to new shake flasks with 100 mL of VMM containing a specific carbon source [2% XG (P-XYGLN, Megazyme) or 2% mannan (P-MANCB, Megazyme) or 2% MLG (P-BGBM, Megazyme) or 2% Starch]. After 4 h in the new carbon source, the mycelia were harvested over a filter, flash frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted for library generation using the standard procedures as described in Tian et al. [15 (link)].
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