Archaeal enzymatic activity was assayed in 1.5 mL of 50 mM phosphate buffer (pH 6.0) containing 15 μg of heat-purified extract and 15 mg pure potato starch (Sigma). For total protein extracted from mini tubers, the activity of enzymes α-amylase, amylopullulanase, and glucoamylase was assayed in reactions containing 1% potato starch and pullulan (Sigma), and 2% maltose (Sigma)/maltotriose (Fisher Scientific), respectively. The reaction was conducted in phosphate buffer at pH 7.0 for starch and pullulan, and pH 5.6 for maltose/maltotriose. The reactions were incubated at 85°C for 30 min and centrifuged at 10,000 × g for 2 min. Total glucose in 500 μL of the hydrolysate was determined by the modified 3, 5-dinitrosalicylic acid (DNS) method (Miller, 1959 (link)). Increase in absorbance due to the release of glucose was measured at OD540. AGPase activity was assayed according to Kulichikhin et al. (2016) (link) based on coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions generating NADPH as the end product. The increase in absorbance due to NADPH was measured at OD340 using a control reaction without the enzyme as a reference.
Potato starch
Manufactured by Merck Group
Sourced in United States, Germany, Poland, Canada
Potato starch is a fine, white, powdery substance derived from the starch-containing cells of potato tubers. It serves as a thickening agent and provides texture in various food and non-food applications.
Automatically generated - may contain errors
Lab products found in correlation
3 5 dinitrosalicylic acid, by Merck Group (6 mentions)
Amylopectin, by Merck Group (5 mentions)
Wheat starch, by Merck Group (4 mentions)
Ascorbic acid, by Merck Group (4 mentions)
Acarbose, by Merck Group (4 mentions)
Sodium hydroxide (naoh), by Merck Group (4 mentions)
Folin ciocalteu reagent, by Merck Group (3 mentions)
Sucrose, by Merck Group (3 mentions)
Gallic acid, by Merck Group (3 mentions)
Glycerol, by Thermo Fisher Scientific (3 mentions)
Polyvinyl alcohol (pva), by Merck Group (3 mentions)
Isomaltotriose, by Merck Group (3 mentions)
Isomaltose, by Merck Group (3 mentions)
Panose, by Merck Group (3 mentions)
Nigerose, by Merck Group (3 mentions)
Isomaltopentaose, by Merck Group (3 mentions)
Acetic acid, by Merck Group (3 mentions)
Sodium potassium tartrate, by Merck Group (3 mentions)
Citric acid, by Merck Group (3 mentions)
Sodium chloride (nacl), by Merck Group (3 mentions)
67 protocols using potato starch
1
Enzymatic Characterization of Archaeal Carbohydrases
2
Starch Hydrolysis by Bm-2 Fungal Strain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study the ability of the Bm-2 strain to hydrolyze soluble potato starch, (Sigma Aldrich, St Luis, MO, USA), Petri plates with YS solid medium were inoculated with a 1 cm-diameter dish of mycelium grown for 5 days, incubated at 32 ± 2 °C, and evaluated (two different plates) every 48 h for 6 days. The plates were flooded with Lugol’s iodine solution (Sigma Aldrich) to monitor the production of a starch degradation halos after iodine staining [30 (link)]. A pale-yellow zone around the mycelium indicated starch degrading activity.
The activity of α-amylase was determined by a modified method, following Ahmed et al. [31 (link)]. The assay mixture consisted of 0.5 mL soluble starch as substrate (1% in 0.1 M sodium acetate buffer pH 5.0) and 0.5 mL of crude extract. After incubation for 20 min at 40 °C, the reaction was stopped by cooling the sample to 4 °C. After cooling, starch hydrolysis was determined through glucose release by the Miller DNS method [32 (link)]. The amount of enzyme production was expressed as U/mL. A unit of enzyme activity was defined as the amount of enzyme that released 1 µg of reducing sugar as glucose standard per minute under assay conditions.
The activity of α-amylase was determined by a modified method, following Ahmed et al. [31 (link)]. The assay mixture consisted of 0.5 mL soluble starch as substrate (1% in 0.1 M sodium acetate buffer pH 5.0) and 0.5 mL of crude extract. After incubation for 20 min at 40 °C, the reaction was stopped by cooling the sample to 4 °C. After cooling, starch hydrolysis was determined through glucose release by the Miller DNS method [32 (link)]. The amount of enzyme production was expressed as U/mL. A unit of enzyme activity was defined as the amount of enzyme that released 1 µg of reducing sugar as glucose standard per minute under assay conditions.
3
Bioinspired Hydrogel Composite Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The materials used were polyvinyl alcohol (PVA, molecular weight (Mw) 85,000–124,000 Da, 99% hydrolyzed, Sigma-Aldrich, Louis, MO, USA), gelatin (Sigma-Aldrich, Louis, MO, USA), sodium carboxymethyl cellulose (NaCMC, Mw ~250,000 Da, Sigma-Aldrich, Louis, MO, USA), potato starch (Sigma-Aldrich, Louis, MO, USA), and manuka honey (Manuka Doctor 20+, New Zealand).
4
Enzymatic Inhibition Assay for Starch Hydrolysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The method described by McDougall et al [10 (link)] was used with minor modifications. Potato starch (Sigma, S2004) was used as the substrate and acarbose as a positive inhibition control. The absorbance data was used to calculate enzyme inhibition rates and IC50 values as follows;
Where, [P]25 = absorbance after 25 min, [P]0 = absorbance at 0 min and δt = change in time. The percentage inhibition in the presence of cinnamon or know inhibitor was calculated as follows;
Where, Rate = rate with inhibitor/sample, Control rate = rate in control. Inhibitor concentrations were plotted against calculated % inhibitions, to produce a linear regression trend line, and the equation for the line used to calculate IC50 values.
Where, [P]25 = absorbance after 25 min, [P]0 = absorbance at 0 min and δt = change in time. The percentage inhibition in the presence of cinnamon or know inhibitor was calculated as follows;
Where, Rate = rate with inhibitor/sample, Control rate = rate in control. Inhibitor concentrations were plotted against calculated % inhibitions, to produce a linear regression trend line, and the equation for the line used to calculate IC50 values.
5
Synthesis and Characterization of Carbogel from Potato Starch
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EXAMPLE 1
To obtain 5 g of a carbogel from potato starch, 25 g of potato starch (Sigma Aldrich) were weighed. Then, 250 g of a potato starch suspension was prepared in proportion of 10% by wt. of starch—90% by wt. of distilled water, and the suspension was placed in a water bath and heated to 75° C. After 30 min from the polycondensation of starch, the obtained product was removed from the water bath and aged for 24 h. Then, the sample was poured over with 96% ethanol (POCh) solution and left for another 24 h tightly sealed. After 6 days, the solvent exchange was repeated. After next 6 days since the last exchange of the alcohol solution, the obtained alcogel was subjected to pyrolysis at a temperature of 700° C. under argon atmosphere (99.999%) for 6 hours.
The obtained carbogel was characterised by an electrical conductance of 0.83 S/cm at a temperature of 25° C. and an electrical conductance activation energy of Ea=0.007 eV. Electrochemical tests showed that obtained material is characterised by a gravimetric capacity—after 40 cycles under a current load of C/2, it amounted to 136 mAh/g.
6
Simulated Gastrointestinal Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pepsin from porcine stomach mucosa (250 U/mg), pancreatin (8 × USP) from porcine pancreas, porcine bile extract, mucin from porcine stomach-type II, albumin, resazurin, cysteine, peptone, yeast extract, pectin, xylan, gum arabic, potato starch, casein, glucose, and inulin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Phenolic compounds standards (2,4 dihydroxybenzoic, 3,4 dihydroxybenzoic, gallic, benzoic, caffeic, ferulic, p-coumaric, salicylic, rosmarinic, and 5-caffeoylquinic acids) were purchased from Sigma-Aldrich Chemical Co. All solvents were HPLC grade from Tedia (Fairfield, OH, USA). HPLC grade water (Milli-Q system, Millipore, Bedford, MA, USA) was used throughout the experiments.
7
Enzymatic Activities of Yeast Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amylase and pectinase activities were performed in a minimal medium using 2% potato starch (Sigma Aldrich, Burlington, MA, USA) and 1% pectin from the citrus peel (Sigma Aldrich, Burlington, MA, USA) as a carbon source, respectively [75 (link)]. Cellulase production was evaluated in Congo red medium with 2% methylcellulose [76 (link)] and protease production in skim milk medium [77 (link)]. All plates were inoculated with 5 µL of yeast solution (1 × 108 cell/mL) and incubated at 28 °C for 5 days. In order to detect amylases, the colony was flooded in an iodine solution (1% w/v). The presence of a clear halo around the colony was considered positive. Pectinase activity was visualized by a clear halo around the colony after it was flooded with a CTAB solution (cetyltrimethylammonium bromide, 5% w/v). Cellulase and protease activities were demonstrated by a clear halo around the colony. The enzymatic index (EI) was determined by subtracting the colony’s diameter from the halo’s diameter [64 (link)]. Assays were performed in triplicate for each strain.
8
Protein Expression in Pichia pastoris
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli DH5α strain (Sangon Biotech, Shanghai) was used for gene cloning and plasmid maintenance throughout the study, and Pichia pastoris X33 strain (Invitrogen, Carlsbad, CA, USA) was used for protein expression. The pGAPZαA vector (Invitrogen, Carlsbad, CA, USA) was used for gene cloning. Potato starch, wheat starch, corn starch, amylose (potato) and amylopectin (potato) were obtained from Sigma-Aldrich (St. Louis, MO). The Mix (Green) for PCR amplification was from TsingKe (Beijing, China). The QIAprep Spin Miniprep Kit and RNeasy@ Midi Kit were obtained from Qiagen (Valencia, CA). SMART® cDNA Library Construction Kit, Advantage® 2 PCR Kit and the Talon metal affinity resin were purchased from Clontech Laboratories, Inc. (Mountain View, CA). The Amicon@ ultra centrifugal filters were obtained from Millipore (Billerica, MA). All other reagents were purchased from general commercial suppliers and used without further purification.
9
SDS-PAGE Analysis of MaAmyB Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE analysis was used to determine protein masses. After refolding the location of the MaAmyB protein could be visualized in the gels by staining for its starch-acting activity. SDS-PAGE51 (link) was performed using precast THX gels (Bio Rad, Veenendaal, the Netherlands). After loading and running, gels were washed 3 times for 5 min in Milli Q water to remove SDS and allow protein refolding, then incubated for 2 h at 37 °C in standard assay buffer containing 0.5% soluble potato starch (Sigma-Aldrich, Zwijndrecht, the Netherlands)52 (link). After incubation the gels were stained with Lugol’s iodine (2.5% I2/5% KI) to visualize protein bands with α-amylase activity. After imaging, gels were partly destained by washing in Milli Q water and then stained with Bio-Safe™ Coomassie Stain (Bio Rad, Veenendaal, the Netherlands) to visualize proteins. The Fermentas PageRuler prestained marker was included in each gel.
10
Saliva Proteomics: Amylase Removal
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Saliva protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). By taking 300 μg of proteins from each individual sample, every four samples were pooled into one sample in cancer group and healthy control group, respectively, thus 5 pool cancer samples and 5 pool healthy control samples were prepared. All the 10 pooled samples were subjected to potato starch affinity column for efficiently removal of alpha amylase as previously described53 (link). Briefly, homemade affinity column packed with potato starch (Sigma Aldrich, Saint Louis, USA) was used to trap amylase and the flow through were collected for further analysis. Equal amount of protein from each pool sample was then used for the following experiment. Two pooled saliva samples were made from all the 10 pooled samples as a GIS for the comparison between two TMT-6plex experiments. 1D SDS-PAGE and 2D-DIGE were run as previously described41 (link) to test the efficiency of amylase removal.
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!