The H2O2 content was analysed as per the method proposed by Patterson et al. [62 (link)]. 0.5 g of fresh sample was crushed in acetone (1 mL) and centrifuged (at 5000× g for 20 min at 4 °C). 20 µL of 20% titanium chloride in conc. HCl was added to the supernatant, followed by addition of ammonium sulphate (200 µL of 17 M, (Himedia, Mumbai, India). The precipitates formed were washed thoroughly with acetone and finally dissolved in 1.5 mL of 2 N H2SO4 (Himedia, Mumbai, India). The absorbance of the sample was measured at 410 nm. The content of H2O2 was computed by taking H2O2 as standard and recorded as μmol g−1 FW. Visualisation of accumulation of H2O2 content in leaves was done by following the protocol stated by Thordal and Christensen [63 (link)] in which leaves were immersed in 3,3′-diaminobenzidine (1%, DAB, Sigma Alrdrich, Mumbai, India) in the dark, followed by decolouration to clearly visualise the brown spots. H2O2 content in B. juncea roots was visualised by staining them with 2′,7′-dichlorofluorescin diacetate (DCF-DA, Sigma Alrdrich, Mumbai, India), following the method given by Rodriguez-Serrano et al. [64 (link)].
Ammonium sulphate
Manufactured by HiMedia
Sourced in India
Ammonium sulphate is a crystalline chemical compound with the formula (NH4)2SO4. It is a common laboratory reagent used in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Peptone, by HiMedia (2 mentions)
Yeast extract, by HiMedia (2 mentions)
Monopotassium phosphate, by HiMedia (2 mentions)
Malt extract, by HiMedia (2 mentions)
Guar gum, by Merck Group (1 mentions)
P nitrophenyl substrate, by Merck Group (1 mentions)
P nitrophenol, by Merck Group (1 mentions)
Locust bean gum, by Merck Group (1 mentions)
Mannose, by Merck Group (1 mentions)
Mannotetraose, by Megazyme (1 mentions)
Urea, by HiMedia (1 mentions)
Dipotassium phosphate, by HiMedia (1 mentions)
Magnesium sulphate, by HiMedia (1 mentions)
Casein, by Sisco Research Laboratories (1 mentions)
Sodium citrate, by HiMedia (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Gallic acid, by Tokyo Chemical Industry (1 mentions)
Temed, by HiMedia (1 mentions)
Sodium dodecyl sulphate (sds), by HiMedia (1 mentions)
Glutamic acid, by HiMedia (1 mentions)
7 protocols using ammonium sulphate
1
Quantitative and Qualitative Analysis of H2O2 in Plants
2
Characterization of Manno-oligosaccharides and Plant Polysaccharides
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Mannobiose (M2), mannotriose (M3) and mannotetraose (M4) were purchased from Megazyme (Bray, Ireland). Locust bean gum (LBG), solka floc, glucose, mannose, guar gum, p-nitrophenyl-substrates, p-nitrophenol and other chemicals were procured from Sigma-Aldrich, USA. Yeast extract, peptone, urea and ammonium sulphate were procured from Hi-Media, India. The palm kernel cake was purchased from M/s Meh Impex, Chennai, India. Food grade konjac gum (glucomannan) was obtained from New Foods, Illinois, USA. Fenugreek seed meal (seed meal of Trigonella foenum-graecum), wheat bran and wheat straw were purchased from local market.
3
Casein and Gelatin Protocol
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Casein was purchased from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Gelatin, pure amino acids (L form), Minimal Davis Broth (Ingredients g/L: Dipotassium phosphate, 7.0 g; Monopotassium phosphate, 2.0 g; Sodium citrate, 0.5 g; Magnesium sulphate, 0.1 g; Ammonium sulphate,1.0 g) and other media components were purchased from Hi-Media Laboratories (Mumbai, India). All other chemicals used were of highest purity grade.
4
Phenolic Substrate Extraction and Characterization
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Phenolic substrates including ferulic acid, gallic acid, syringaldehyde (SGA) and catechol were purchased from Tokyo Chemical Industry Co., Ltd. 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and Coomassie Brilliant Blue G-250 were obtained from Sigma-Aldrich. DEAE cellulose and Sephacryl (S-100) were acquired from Bio-Rad and Pharmacia, respectively. Basic chemicals, namely malt extract, ferrous sulphate, acrylamide, bis-acrylamide, ammonium persulphate, TEMED (N,N,N′,N′-tetramethyl ethylenediamine), sodium dodecyl sulphate (SDS), Tris buffer, ammonium sulphate, glutamic acid, monopotassium phosphate, copper and zinc sulphate, were obtained from Himedia. The centrifugal concentrators were obtained from Amicon Millipore, while the dialysis tubing was from Thermo Fischer Scientific. The bundle of human hair was picked up from a local salon. The collected hairs belonged to the same person. All other chemicals used were of analytical grade.
5
Colloidal Chitin Minimal Salts Broth Preparation
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Ammonium sulphate, 3,5-dinitrosalicylic acid (DNS), powdered shrimp chitin, yeast extract powder, malt extract powder and other ingredients used in the preparation of colloidal chitin minimal salts (CCMS) broth were purchased from HiMedia laboratories Pvt. Ltd., Mumbai, India. Laminarin derived from Laminaria digitata and 12 kDa cutoff dialysis tubing were procured from Sigma-Aldrich chemicals Pvt. Ltd., Bengaluru, India. Concentrated hydrochloric acid was procured from Merck life science Pvt. Ltd., Mumbai, India. Carboxymethyl cellulose (CMC) was procured from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. An actinomycete strain AFM-1 isolated from marine sediment sample from Udupi district, Karnataka, India was used in this study. AFM-1 was identified as Streptomyces rimosus based on phenotypic and 16SrDNA sequencing by National Chemical Laboratory (NCL), Pune, India (GenBank accession No. MT604984). Tinopal CBS-X (generic) and 280 g per square meter (GSM) 65/35 polyester-cotton blend laboratory coat clothing material (LCCM) were procured from the local market. Erma ESM11 stage micrometer and eye-piece graticule were procured from Erma Inc., Tokyo, Japan.
6
Optimization of Amylase Production
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OFAT method was carried out by changing parameters of factors in a sequential manner. The factors considered for optimization were pH of the media, Incubation temperature, Carbon and nitrogen sources and presence of metal ions. The pH of the media was adjusted between 4 and 10. The incubation temperature ranged between 4 °C and 45 °C. Additional carbon sources like glucose, fructose, sucrose, dextrose, inositol, mannitol, rhamnose, maltose, lactose, trehalose, sodium citrate, arabinose (HiMedia Laboratories, Mumbai, India, LR grade) was supplemented at 10 g/L along with Starch. The effect of nitrogen source on amylase production was evaluated by replacing peptone with malt extract, beef extract, yeast extract, peptone, casitone, corn steep liquor (HiMedia Laboratories, Mumbai, India, LR grade) and inorganic nitrogen sources such as ammonium sulphate, ammonium chloride, ammonium carbonate, potassium nitrate and sodium nitrate (HiMedia Laboratories, Mumbai, India, LR grade). Various metal ions such as Fe2+, Cu2+, Mg2+, K+, Ca2+, Mn2+, Ba2+, Sn2+, Zn2+, Co2+, Ni2+at 0.5 g/L were supplemented in the media in the form of metal salts. All the experiments were performed in triplicates and the data is represented in Mean ± Standard deviation.
7
Ammonium Sulphate Precipitation of Protease Inhibitors
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The crude PI samples were purified using standard Ammonium sulphate precipitation. Ammonium sulphate (Hi Media) required to precipitate the protease inhibitor was optimized using 30% saturation to the crude extract. After complete dissolution of Ammonium sulphate, the solution was kept for precipitation at 4°C for 4 h. Protein precipitated was collected by centrifugation at 10,000rpm for 15 min at 4°C. The precipitate obtained after Ammonium sulphate precipitation was further dialyzed against 0.01M phosphate buffer (pH 7.5), in order to remove the Ammonium sulphate from the precipitate. Purified fractions were finally screened for protease inhibitor assay.
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