Bromophenol blue sodium salt

Methanol (MeOH, >99%), (GVL, 99%), 2H-methylimidazole (MIM, 97%), Xylenol Orange disodium salt (XO), Bromophenol Blue sodium salt (BB), Congo red (CR) and zinc nitrate hexahydrate (Zn(NO₃)₂∙6H₂O, 99%), and deuterium chloride (DCl (35%)/D₂O) were purchased from Sigma Aldrich (Darmstadt, Germany); deuterated dimetyhlsulphoxide (DMSO-d6) was purchased from Eurisotop (Saint-Aubin, France) and was used without further purification.

Protein fractions with antibacterial activity were analyzed by SDS-PAGE electrophoresis. DL-dithiothreitol, acrylamide/bis-acrylamide (30% solution), bromophenol blue sodium salt (Sigma-Aldrich, Schnelldorf, Germany), N, N, N′, N′-tetramethylethylenediamine (TEMED), ammonium persulphate (APS) (GE Healthcare, Stockholm, Sweden), and Laemmli sample buffer (2×), for SDS PAGE (SERVA, Heidelberg, Germany), were used for electrophoreses analysis. Because commercial Laemmli buffer does not contain any reduction reagent, 10 mM DTT were added as a reducing sample buffer (concentrations refer to 1× sample buffer). Equal volumes containing approximately 25 μg/lane of the samples dissolved in Laemmli sample buffer and protein standard mixture (Precision Plus Protein™, All Blue, Bio-Rad, Feldkirchen, Germany) were separated by 12.5% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G-250.

Protein fractions from R. venosa hemolymph were analyzed by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE) with the molecular weight marker ranging from 250 kDa to 10 kDa using a 5% stacking gel and 12.0% resolving gel, according to the Laemmli method with modifications [64 (link)]. DL-dithiothreitol, acrylamide/bis-acrylamide (30% solution), bromophenol blue sodium salt (Sigma-Aldrich, Schnelldorf, Germany), N, N, N′, N′-tetramethylethylenediamine (TEMED), ammonium persulphate (APS) (GE Healthcare, Stockholm, Sweden), and Laemmli sample buffer (2×), for SDS PAGE (SERVA, Heidelberg, Germany), were used for electrophoresis analysis. Equal volumes containing approximately 20 μg/lane of the samples dissolved in Laemmli sample buffer and protein standard mixture (Precision Plus Protein™, All Blue, Bio-Rad, Feldkirchen, Germany) were applied on 12.0% SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G-250 (Bio-Rad Laboratories GmbH, Germany).

Sodium dihydrogen phosphate and hydrogen peroxide were acquired from Merck (Darmstadt, Germany) and fluorescein, potassium phosphate dibasic, AAPH (2,20-azo-bis-(2-methylpropionamidine)-dihydrochloride), trolox (6-hydroxy-2,5,7,8-tetramethylbroman-2-carboxylic acid), DNA (deoxyribonucleic acid salt) from calf thymus, bromophenol blue sodium salt, and phenazine methosulfate solution (PMS) were purchased from Sigma Chemical (St. Louis, MO, USA). Agarose and GreenSafe Premium were purchased from Nztech (Lisboa, Portugal). Iron(III) chloride (FeCl₃) was acquired from Panreac (Barcelona, Spain) and Tris-Acetate EDTA (ethylenediaminetetraacetic) (TAE) buffer was purchased from Grisp (Porto, Portugal). Caucasian colon carcinoma (Caco-2) and mucous producing human colon (HT29-MTX-E12) cells were obtained from the European Collection of Authenticated Cells Cultures (ECACC 8601020 and 12040401, respectively) through Sigma-Aldrich (St. Louis, MO, USA; ECACC) (references 09042001 and 12040401, correspondingly). DMEM (Dulbecco’s Modified Eagle’s Medium), Pen-Strep, and non-essential amino acids 100× were purchased from Lonza (Basel, Switzerland) and Fetal Bovine Serum (FBS) was obtained from Biowest (Nuaillé, France).

Chlamys farreri were purchased from a local market (Dalian, Liaoning, China). Ethylene diamine tetraaceticaciddisodiumsalt (Na₂EDTA), sodium dihydrogen phosphate, sodium hydrogen phosphate, DTNB, bromophenol blue sodium salt (BPB), β-mercaptoethanol, acrylamide, N, N′-Methylene-bis (acrylamide), Tricine, G-250 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents used in this study were analytical and HPLC grade.