Binding studies to assess the ability of DmCE1A_CBM48 and DmCE1B_CBM48 to bind insoluble polysaccharides were performed using pull-down studies as described in Kmezik et al. (32 (link)), except 50 mM sodium phosphate buffer (pH 6.5) was used as the buffer, on the insoluble polysaccharides: ivory nut mannan (Carbosynth), cellulose (Merck), birch xylan (Merck), beech xylan (Merck), mixed-linkage barley glucan (Megazyme), and potato starch (Merck). All polysaccharides were washed three times in the aforementioned buffer before being used in the assay. Binding studies using soluble polysaccharides by affinity gel electrophoresis were performed as previously described (67 , 68 (link)) using carboxymethylcellulose, galactomannan (Megazyme), glucomannan (Megazyme), wheat arabinoxylan (Megazyme), and xyloglucan (Megazyme). All polysaccharides were used at a concentration of 0.5% w/v in the polyacrylamide gels.
Birch xylan
Manufactured by Merck Group
Sourced in Germany
Birch xylan is a hemicellulose extracted from the wood of birch trees. It serves as a structural component in the cell walls of plants. Birch xylan is commonly used as a reagent or additive in various laboratory and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Potato starch, by Merck Group (2 mentions)
Beech xylan, by Merck Group (2 mentions)
Glucomannan, by Megazyme (1 mentions)
Galactomannan, by Megazyme (1 mentions)
Xyloglucan, by Megazyme (1 mentions)
Wheat arabinoxylan, by Megazyme (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Xylose, by Megazyme (1 mentions)
Xylobiose, by Megazyme (1 mentions)
Xylotriose, by Megazyme (1 mentions)
Xylotetraose, by Megazyme (1 mentions)
Apple pectin, by Merck Group (1 mentions)
Porcine mucin, by Merck Group (1 mentions)
Carboxymethylcellulose, by Merck Group (1 mentions)
5 protocols using birch xylan
1
Binding Studies of Carbohydrate-Binding Modules
2
Binding of Bo_M to Insoluble Polysaccharides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Assays to evaluate the ability of Bo_M to bind to insoluble polysaccharides were performed on the insoluble fractions of ivory nut mannan (Carbosynth), cellulose (Merck), birch xylan (Merck), beech xylan (Merck), mixed-linkage barley glucan (Megazyme) and potato starch (Merck). 2.5% w/v solutions of insoluble polysaccharides, suspended in 50 mM tris(hydroxymethyl)aminomethane pH 8.0, 250 mM NaCl, 5% glycerol, were incubated with Bo_M (0.1 g/L) at 37 °C for 30 min (incubation step sample). The insoluble polysaccharides were collected by centrifugation (14,000 rpm, 5 min) before being washed with buffer and incubated for an additional 10 min (wash sample). The insoluble polysaccharides were collected by centrifugation once more and washed with 8 M urea to solubilize any bound protein (elution sample). SDS-PAGE and ImageLab (Bio-Rad) were used to visualize Bo_M and quantify its concentration. BSA was used as control and, similar to previous reports [21 (link)], did not bind to any of the tested substrates.
3
Birch Xylan Hydrolysis Product Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the analysis of the hydrolysis products of birch xylan (Sigma Aldrich, Steinheim, Germany), XOS: xylose, xylobiose, xylotriose, xylotetraose, xylopentose, and xylohexose (Megazyme, Wicklow, Ireland) as solutions of 0.1% were used as standards. The 0.1% xylan (50 mM citrate buffer pH 4.8) was analyzed after applying 1 μg xylanase I or xylanase II, respectively, at 30°C. Aliquots from these incubation were taken after 15 min, 60 min, and 17 h, spotted and analyzed on silica-gel G-60 plates F254 (20 × 20 cm, Merck). For separation, ethyl acetate/acetic acid/formic acid/distilled water (9:3:1:4; v/v/v/v) was used as mobile phase. After separation, the plates were dried at 80°C for 10 min followed by visualization of the sugars by spraying freshly prepared 0.2% (w/v) orcinol in sulfuric acid/methanol (1:9; v/v) solution and incubation at 80°C for 2 h.
4
Defined Growth Medium for Anaerobic Microbiome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The defined growth medium was prepared in a 0.05 M potassium phosphate buffer made from 1 M stock solutions (ml/L): K2HPO4 (28.9) and KH2PO4 (21.1); mineral salts (mg/L): MgSO4∗7H2O (36), FeSO4∗7H2O (0.1), CaCl2 (9), MnSO4∗H2O (3), ZnSO4∗7H2O (1), CoSO4∗7H2O (1), CuSO4∗5H2O (1), (NH4)6Mo7O24∗4H2O (1), NaCl (527); hemin (5 mg/L); vitamin K1 (0.5 mg/L); L-amino acids (g/L): Ala (0.044), Arg (0.023), Asn (0.038), Asp (0.038), Glu (0.036), Gln (0.018), Gly (0.032), His (0.027), Ile (0.060), Leu (0.120), Lys-HCl (0.080), Met (0.023), Phe (0.050), Pro (0.041), Ser (0.095), Thr (0.041), Trp (0.009), Val (0.060), Tyr (0.015); vitamins (mg/L): biotin (0.25), Ca-pantothenate (0.25), folic acid (0.25), nicotinamide (0.25), pyridoxine-HCl (0.50), riboflavin (0.25), thiamine-HCl (0.25) and other components (g/L): bile salts (0.5), NaHCO3 (2.0), Tween-80 (0.5), Na-thioglycolate (0.5), and Cys-HCl (0.5, freshly made). The carbohydrate substrates were sterilized separately and mixed with the medium before cultivation. Two substrate combinations, either birch xylan (Sigma-Aldrich, United States) or apple pectin (Sigma-Aldrich, United States) with porcine mucin (Type II, Sigma Aldrich, United States), were added to the base medium in equal amounts (2.5 g/L each). The pH of the growth medium was 7.2 ± 0.1.
5
Enzymatic Assays for Xylanase and Cellulase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The xylanolytic and cellulolytic activity in the enzymatic extracts was determined by the dinitrosalicylic acid method [107 (link)]. The xylanase and cellulase activities were determined by incubating the enzyme extract at 50 °C and using 0.5% (w/v) birch xylan (Sigma-Aldrich, Darmstadt, Germany) and 0.5% (w/v) carboxymethylcellulose (Sigma-Aldrich, Darmstadt, Germany) as substrates in 100 mM acetate buffer (pH 5.3). A unit (U) of xylanase/cellulase was defined as the amount of enzyme required to release 1 μmol of xylose equivalent/glucose per minute under the conditions tested.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!