Bromophenol blue

Structural polypeptides from the purified OBs and occlusion derived virions (ODVs) were analyzed on 11% SDS-polyacrylamide slab gels (SDS-PAGE). ODVs were harvested from OBs by mixing 10 μl of purified OBs at 10¹⁰ OBs/ml with an equal volume of 0.1 M Na₂CO₃ and incubating at 28°C for 30 min. Undissolved OBs and other debris were pelleted at 6,000 x g for 5 min. A 10 μl volume of purified OBs at a concentration of 10¹⁰ OBs/ml and 10 μl of harvested ODVs were solubilized with an equal volume of 2x Laemmli sample buffer (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% bromophenol blue, BioRad) by heating to 100°C for 5 min prior to electrophoresis. Electrophoresis was performed at 50 mA during 2 h. Finally, the gels were stained in Comassie Brilliant Blue R solution (0.1% Comassie R Brilliant Blue, 10% v/v acetic acid and 50% v/v ethanol) for 30 min and destained with a bleaching solution (9.45% v/v ethanol and 6.75% v/v acetic acid). The polypeptide profiles were visually inspected and photographed.

Nine samples containing 5 μg of native TeNT (nTeNT) and irradiated TeNT 1 - 8 kGy
(iTeNT) were added in 15 μL of reducing sample buffer 0.0625 M Tris (Synth)-HCl
(VETEC), 2% SDS (Synth), 10% Glycerol (VETEC), 5% 2-Mercaptoethanol (Merck), 1M
Urea, 5% Bromophenol Blue (Bio-Rad) or non-reducing buffer, with the same
composition as above, excepted for the 2-Mercaptoethanol which was ommited,
heated at 100 ºC for 5 minutes and applied to the gel. Six microliters of
prestained protein standard (Bio-Rad) was loaded in each gel.
The electrophoretic mobility analysis (SDS PAGE), in a discontinuous and
denaturant system was performed according to Laemmli [37 (link)] in Mini-Protean IV system (Bio-Rad). The stacking gel
was prepared at a concentration of 4% and the resolving gel at a concentration
of 7.5%, both are composed of acrylamide (Sigma Adrich)/bis-acrylamide (Merck).
Electrophoretic migration was performed for approximately two hours (80 volts -
20-30 mA) in a running buffer solution [0.025 M Tris (Synth)-0.192M Glycine
(Synth) pH 8.3]. The gels were stained with Coomassie Blue R250 [50% Methanol
(Synth), 10% Acetic Acid (Synth), 0.1% Coomassie Blue R 250 (Bio-Rad)].

Cell lysis and extraction of the cytoplasmic and nuclear fractions was performed using the NE-PER™ Nuclear and Cytoplasmic Extraction kit (Thermo Scientific, 78835) according to the manufacturer’s instructions. The resulting fractions were then mixed with 5× Laemmli buffer (62.5 mM Tris–HCl, pH 6.8 (Sigma, T6066), 2% sodium dodecyl sulfate (Sigma, L4509), 10% glycerol (Sigma, G5516), 5% beta-mercaptoethanol (Sigma, 63689), 0.02% bromophenol blue (Bio Rad, 1610404)) and separated by SDS-polyacrylamide gel electrophoresis.

A total of 20 g of protein was present in an aliquot of the skimmed milk, which was then combined (1:1) with a sample buffer at pH 6.8 made up of 62.5 mM Tris-HCl, 25% glycerol, 4% SDS, and 0.01% bromophenol blue (Bio-Rad). After that, it was run through a 7.5% native SDS-PAGE (Minigel, Bio-Rad, California, USA) with 0.1% bovine gelatin (Sigma-Aldrich, Saint Louis, MO, USA) in the resolving gel. The gels were then incubated at 37°C for an overnight period in a 50 mM Tris-base buffer (Bio-Rad, California, USA) with pH 7.4 and 200 mM NaCl, 0.02% Brij-35, and 5 mM CaCl2 for gelatinolysis after 30 min at room temperature (25°C) in a renaturing solution of 2.5% (v/v) Triton X-100. The gels were stained for 30 min with 0.5% Coomassie Blue R-250 (Sigma-Aldrich) in 40% methanol, 10% glacial acetic acid, and 50% distilled water the following day. Next, they were destained for 30 min in 30% methanol, 7.5% glacial acetic acid, and 62.5% distilled water. Later, they were preserved in distilled water. Matrix metalloproteinase-2 and -9 were visible bands against the blue backdrop. After scanning with a CanoScan LiDE 120 Scanner (Canon Inc., Tokyo, Japan), it was integrated using ImageJ software version 1.80 (National Institute of Mental Health, Bethesda, Maryland, USA) [22 (link)]. The area of the band was used to represent the degree of gelatinase.

Components for culture media, tryptone, and yeast extract were purchased from Difco Laboratories, India. Glycine, phenylmethylsulfonyl fluoride (PMSF), isopropyl β-D-1-thiogalactopyranoside (IPTG), Tris buffer and sodium dodecyl sulfate (SDS) were from Amresco, United States. Glucose and NaCl were purchased from Qualigen, India. Low molecular weight marker for SDS-PAGE was from GE Healthcare, United States. Dithiothreitol (DTT), acrylamide, bis-acrylamide, urea, ammonium persulfate (APS) and Proteinase K were purchased from Sigma-Aldrich, United States. Bromophenol blue, Tetramethylethylenediamine (TEMED), and Ethylenediaminetetraacetic acid (EDTA) were procured from BIO-RAD, United States.

The peptide fraction extracted from H. illucens larvae infected with E. coli, M. flavus and from uninfected larvae (control) was fractionated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In detail, at 15 µL for each protein extract, the loading buffer 1X, composed of 2% SDS (Bio-Rad, Hercules, CA, USA), 50 mM TRIS-HCl pH 6.8 (Merck Millipore, Burlington, MA, USA), 10% Glycerol (Merck Millipore, Burlington, MA, USA) and bromophenol blue (Bio-Rad, Hercules, CA, USA), was added, and they were separated on a 20% SDS-PAGE gel. After the run, the gel was stained with GelCode™ Blue Safe Protein Stain (Thermo Fisher Scientific, Waltham, MA, USA) and destained with Milli-Q water. A total of 3 bands for each condition (E. coli, M. flavus, control) were cut and in situ hydrolyzed with trypsin as previously described [56 (link)]. Peptide mixtures were extracted in 0.2% formic acid (HCOOH) (Merck Millipore, Burlington, MA, USA) and acetonitrile (ACN) (Merck Millipore, Burlington, MA, USA) and vacuum dried via a SpeedVac System (Thermo Fisher Scientific, Waltham, MA, USA).

Sodium dodecyl sulphate (SDS), β-mercaptoethanol, Tween 20, 15% Criterion Tris-HCl polyacrylamide precast gels, bromophenol blue, non-fat dry milk, Tris-buffered saline (TBS) were purchased from Bio-Rad Laboratories. NaCl, Nonidet P-40, sodium deoxycholate, Tris-HCl, PBS, Dulbecco’s PBS (D-PBS), N-Lauroylsarcosine sodium salt solution (sarkosyl NL), PMSF, PK, sucrose, Kodak Biomax MR and XAR films were ordered from MilliporeSigma; Odyssey Blocking Buffer from LI-COR Biosciences; methanol, GdnHCl solution from Thermo Fisher Scientific Inc.; EDTA from Promega; polyvinylidene difluoride (PVDF) membrane (Immobilon-P or Immobilon-FL) from EMD Millipore and ECL and ECL plus reagents from GE Healthcare Life Sciences. Antibodies used were primary mouse mAb 3F4 (to human PrP residues 106–110), Tohoku-2 (to human PrP residues 97–103),^{38 (link)} GFAP Ab and Microglia Ab Iba1 (Biocare Medical), secondary antibodies IRDye 680RD goat anti-rabbit IgG (LI-COR Biosciences) and sheep anti-mouse IgG, HRP-linked whole antibodies from GE Healthcare, Life Sciences.