Cells were sedimented from 50 ml cultures at 1×107 cells ml−1, frozen, resuspended in 300 µl of 1×SSC (0.15 M NaCl, 0.015 M Trisodium citrate) and 100 µl 4×gel sample loading buffer added (0.25 M Tris-HCl, pH 6.8, 40% V/V glycerol, 12% W/V SDS, 20% V/V β-mercaptoethanol, 0.4% W/V bromophenol blue). The mixture was placed in a boiling water bath for 5 minutes and then sedimented for 2 min (2000 rpm, in an Eppendorf 5415C microfuge). For SDS-PAGE, 6 µl of the supernatant was loaded per lane on 10 well, 12% (W/V) polyacrylamide gels with 4% stacking gels and electrophoresed in Tris-Glycine SDS buffer using the MiniProtean II system (BioRad, Hemel Hempsted, UK). Proteins were electroblotted onto Hybond ECL nitrocellulose (GE Healthcare) and incubated with antibody as previously described [69] . An affinity-purified polyclonal antibody raised in rabbits against peptide VAAWKDEDGDWYETG (Eurogentec, Belgium) was used to detect PDS. The primary antibody was detected with an anti-rabbit IgG-alkaline phosphatase conjugate (Sigma-Aldrich, Poole, UK) and visualised using 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium (NBT/BCIP) reagent (Sigma-Aldrich).
Bcip nbt reagent
Manufactured by Merck Group
Sourced in United Kingdom, United States
BCIP/NBT reagent is a chromogenic substrate used for the detection and visualization of alkaline phosphatase enzyme activity in various biological assays and immunohistochemistry applications. It produces a dark purple-blue insoluble precipitate at the site of enzyme activity.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (3 mentions)
Mini protean 2 system, by Bio-Rad (1 mentions)
Anti rabbit igg alkaline phosphatase conjugate, by Merck Group (1 mentions)
Hybond ecl nitrocellulose, by GE Healthcare (1 mentions)
Hybond, by Cytiva (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Streptavidin alkaline phosphatase conjugate, by Thermo Fisher Scientific (1 mentions)
Nap blocker, by G Biosciences (1 mentions)
Donkey anti goat igg, by R&D Systems (1 mentions)
Mab936, by R&D Systems (1 mentions)
Pre stained molecular mass markers, by Bio-Rad (1 mentions)
Mab910, by R&D Systems (1 mentions)
Tmb substrate, by BD (1 mentions)
Varioskan, by Thermo Fisher Scientific (1 mentions)
Immobilon p plates, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Goat anti rabbit secondary antibody, by Merck Group (1 mentions)
8 protocols using bcip nbt reagent
1
Protein Detection via Western Blot
2
Western Blot Analysis of Dectin-1 Protein
3
Quantifying Matrix Metalloproteinases by Western Blot
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According to the Laemmli method, all samples (10 μg of protein) were electrophoresed on SDS-polyacrylamide gel (10%) [29 (link)], blotted to nitrocellulose membranes (Sigma-Aldrich; Saint Louis, MO, USA) at 100 mA for 1 h. The membranes were blocked using 5% (w/v) nonfat powdered milk in the solution of TBS-T (20 mM Tris/HCl buffer, pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20) for 1 h. Then, samples were incubated overnight at 4 °C with antibodies against metalloproteinase-2 (Cat#MAB9021; R&D Systems, USA) or, respectively, directed against metalloproteinase-9 (Cat# MAB936; R&D Systems; USA) in TBS-T which contained 1% bovine serum albumin (w/v). In the next step, several washes in TBS-T buffer were performed. Bounded antibodies were detected using alkaline phosphatase (ALP) coupled with the appropriate antibody in the same solution at room temperature for 1 h with moderate shaking, and then BCIP/NBT reagent was used (Cat# B1911; Sigma; USA). Pre-stained molecular mass markers were used to determine the molecular mass of matrix metalloproteinases (BioRad, Hercules, CA, USA). Representative blots were demonstrated.
4
Western Blot Analysis of Stromelysins
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The 20 μg samples of protein of stromelysin-1 and 20 μg of protein for stromelysin-2 of examined tissue specimens were electrophoresed on 10% SDS-polyacrylamide gel using the method of Laemmli [18 (link)] in non-reducing and reducing conditions and blotted to nitrocellulose membranes (Sigma-Aldrich; Saint Louis, MO, USA) at 100 mA for 1 h. Then, 5% (w/v) nonfat powdered milk in TBS-T solution (20 mM Tris/HCl buffer, pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20) was used for 1 h to block the membranes. Subsequently, they were incubated overnight at 4 °C with a specific antibody directed against human stromelisin-1 (cat. no. MAB905; R&D Systems, Minneapolis, MN, USA) and specific antibody against stromelysin-2 (cat. no. MAB910; R&D Systems, USA) in TBS-T, containing 1% bovine serum albumin (w/v). Following several washes, bounded antibodies were detected using alkaline phosphatase-conjugated with respective secondary antibody in the same solution for 1 h at room temperature with gentle mixing and the next BCIP/NBT reagent (catalogue number B1911; Sigma). The estimation of molecular mass of stromelysines was carried out by means of pre-stained molecular mass markers (BioRad, Berkeley, CA, USA).
5
Quantification of NIP-specific IgG1 and IgM
6
Recombinant Protein Expression and Purification
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The expression and purification of recombinant proteins were monitored by SDS-PAGE electrophoresis and Western blot assays. Culture samples before induction (T0) and after induction of expression (T2), as well as samples of the various purification stages were mixed with protein loading buffer and then denatured at 95 °C for 5 min and loaded onto a 12 % SDS-PAGE gradient gel. Protein observation was performed by staining the gel with Coomasie brilliant blue. For the Western blot analysis, the proteins in the gel without staining were transferred to a polyvinylidene fluoride membrane (Merck Millipore, Massachusetts - USA) in electro-transfer buffer (25 mM Tris, 250 mM Glycine, 20 % Methanol) for 30 min at 100 V, then blocked overnight with 5 % skim milk in Tris-Buffered Saline with 0.05 % Tween 20 (TBS-T). For FHA and cPknF detection, monoclonal anti-His-Tag antibody produced in mouse (GE Healthcare) diluted at 1:20000 were used as primary antibody, followed by membrane incubation for 2 h. Afterward five washes with TBS-T, the membrane was incubated with the secondary antibody Anti-Mouse IgG (whole molecule) −Alkaline Phosphatase produced in goat (Sigma Aldrich, EUA) diluted 1:30 000 for 1 h. The membrane was revelated after previous washing with TBS-T and then adding the substrate for alkaline phosphatase BCIP/NBT reagent (Sigma-Aldrich, EUA).
7
Quantification of MT1-MMP Protein Expression
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Aliquots of tissue extracts (normalized to 20 μg of protein) were subjected to 10% SDS-polyacrylamide gels. After electrophoresis, separated proteins were transferred on to nitrocellulose membranes (Sigma-Aldrich, USA). Non-specific binding sites were blocked with 5% non-fat milk in TBS-T (20 mmol/L Tris-HCl buffer (pH 7.4); 150 mmol/L NaCl; 0.05% Tween-20) for 1 hour. The membranes were incubated with primary anti-human MT1-MMP antibody (R&D Systems, USA) solution overnight at 4°C, and subsequently washed in TBS-T. The membranes were then exposed to the secondary antibody conjugated to alkaline phosphatase (goat anti-mouse IgG; Sigma-Aldrich, 1:2000), for 1 hour at room temperature. The bands were visualized using BCIP/NBT reagent (Sigma-Aldrich). The molecular mass of MT1-MMP was estimated according to the molecular weight markers (Bio-Rad, USA). Western blots were scanned, and densitometric analysis of bands was carried out using ImageJ software. The abundance of MT1-MMP was normalized to the total amount of protein in the sample, and the control group was set as 100%. The amount of protein in the sample was determined using Ponceau S staining.24
8
Detecting MMPs in Tissue Extracts
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The samples of tissue extracts were electrophoresed on 10% SDS-polyacrylamide gel according to the method of Laemmli [16] in nonreducing and reducing conditions. Western blot was performed according to a previously described procedure [17] . The following primary antibodies were used to detect MMPs: mouse monoclonal antibody for human MMP-1 (cat. No. MAB901; R&D Systems; 1: 200) and rabbit polyclonal antibody for human MMP-13 (cat. No. sc-30073; Santa Cruz Biotechnology; 1: 1,000). Bound antibodies were detected using alkaline phosphatase conjugated with goat anti-mouse (cat. No. A2429; Sigma) or goat anti-rabbit secondary antibody (cat. No. A9919; Sigma) and next BCIP/NBT reagent (cat. No. B1911; Sigma).
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