Nupage novex system

Cathepsin B, L and D activities were assessed fluorometrically using the following substrates: Z-Arg-Arg-AMC (Bachem, Bulbendorf, Switzerland), Z-Phe-Arg-AMC (Bachem) and 7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-DNP-D-Arg-amide, as described previously [32 (link)]. LAL was determined by using the pro-fluorescent substrate 4-methylumbelliferyl oleate [33 (link)]. Western blotting was used to assess changes in the protein expression of cathepsin B, L and LAMP-1, following lysis of the cells in water at 4°C, and electrophoresis (90 min, 130 V) using 4–12% bis-tris gels (Novex Nupage system, Life Technologies, Carlsbad, CA, USA) with protein transfer (20 V, 7 min) to a PDVF membrane (iBlot 2, Life Technologies). And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution). Proteins were visualized using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA), following exposure of membranes to chemiluminescence reagents (Western Lightening Plus-ECL, Perkin Elmer, Waltham, MA, USA), with densitometry performed using ImageJ software (National Institutes of Health, USA).

Total protein was extracted using the RIPA buffer (Pierce, Rockford, IL) in the presence of a Protease Inhibitor Cocktail (Pierce). After quantified by the bicinchoninic acid method (Waltham, MA), 80 μg protein was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) using the Novex NuPAGE system (Invitrogen) and denatured for 5 min. Subsequently, the protein was transferred to 0.45‐Am nitrocellulose membranes. Membranes were blocked in TBS‐T buffer containing 5% nonfat milk for 1 h and incubated overnight with the following primary antibodies rabbit anti‐human DLGAP1 and GAPDH (1:800; Abcam, Cambridge, MA). After washed using TBST, the membranes were then hybridized with the horseradish peroxidase (HRP)‐linked antibody goat anti‐rabbit IgG (1:2000; Abcam) for 1 h. Signal detection was carried out with an ECL system (Amersham Pharmacia, Piscataway, NJ).

The levels of expression of pericentrin were determined by Western blot analysis using a rabbit pericentrin polyclonal antibody (ab4448, Abcam Ltd., Cambridge, UK) and the beta actin (A2066, Sigma-Aldrich, Inc., St. Louis, MO, USA) for normalization. The cells washed with PBS buffer plus 0.1 mM Na3VO4 were pelleted and lysed in Laemmli buffer (0.125 M Tris–HCl pH 6.8, 5% SDS) containing protease inhibitors. Lysates were boiled for 2 min, sonicated, and quantitated by Bradford assay. Aliquots containing 30 mg/mL of protein plus 5% b-mercaptoethanol were size fractionated on 3–8% Tris–acetate gel, using the NuPage Novex system from Invitrogen (Carlsbad, CA, USA). After incubation with a peroxidase-conjugated secondary antibody, the immunoreactive bands were visualized by ECL Supersignal on autoradiographic films.

Binding of purified chimeric human IgE and IgG1 anti-BLG antibodies clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 was done by using the BLG ELISA as described above. For detection, however, 1∶1,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) and 1∶5,000 diluted HRP-conjugated goat anti-human IgG-specific antibodies (Jackson ImmunoResearch) were applied.
Bovine BLG (120 µg/480 µl non-reducing sample buffer) was loaded and electrophoresed in a one well pre-casted 4–12% BisTris gel/MOPS running buffer NuPage Novex system (Invitrogen). Then, bovine BLG was electro-blotted onto a polyvinylidene fluoride transfer membrane (Millipore). After blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at RT, this membrane was incubated with 1 µg/ml purified chimeric human IgE anti-BLG antibody clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 for 1 hour at RT. After washing in PBS/0.05% Tween 20, binding of these chimeric human IgE anti-BLG antibodies was determined with 1∶4,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) for 1 hour at RT, followed by a ready-to-use solution of TMB substrate (Sigma) for colorimetric detection.