Amersham hybond ecl nitrocellulose membrane

Cells were lysed in RIPA buffer + 1X PMSF (Alpha Diagnostics). 20μg protein lysates were run on 4%-12% NuPage Bis-Tris Gel (Novex) and transferred onto Amersham Hybond ECL Nitrocellulose Membrane (GE Healthcare). Membranes were blocked in 5% Amersham ECL Prime Blocking Agent (GE Healthcare) + 0.1% Tween 20 (Sigma) in PBS. Membranes were incubated in primary antibody overnight at 4°C, washed 3 times in PBS + 0.1% Tween 20, incubated in HRP-conjugated secondary antibody for 1 h at room temperature and washed 3 times in PBS + 0.1% Tween 20. The membrane was incubated in Amersham ECL Western Blotting Detection Reagent (GE Healthcare) or Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare), depending on the expected strength of signal. The membranes were used to expose Amersham Hyperfilm ECL (GE Healthcare), and films were developed using a Konica SRX-101A Medical Film Processor.

The brain protein lysates such as cortex, hippocampus, and cerebellum were collected from both wild type and reeler mutants (n = 3) mixed with 0.2% DDM lysis buffer consisting of (6× Laemmli buffer containing 100 mM Tris–HCl (pH 6.8), 4% SDS, 60% glycerol, 0.2% bromophenol blue and 10 mM DTT in dH2O). Proteins of the cell lysates were separated by12% SDS-PAGE gels with constant voltage of 110 V for 75 min and transferred to Amersham Hybond ECL nitrocellulose membrane (RPN2032D, GE Healthcare, Germany). After blocking with 5% skimmed milk powder in TTBS (150 mM NaCl, 10 mM Tris and 0.025% Tween® 20 in dH2O), the membrane was incubated with rabbit anti-SERT and anti-ß-actin primary antibodies in TTBS or blocking buffer over night at 4°C. After three washing steps with TTBS the membrane was incubated with rabbit fluorescent infra-red dye-conjugated antibodies (800 and 680nm) for 1h at RT. The membrane was washed for three times in TTBS and immunoreactive proteins were visualized with the LI-COR Odyssey® imaging system Germany.

If not indicated otherwise, supplies were obtained from Sigma (Taufkirchen, Germany). The cell-seeded spider silk scaffolds and negative controls were lysed for blotting in 1% (vol/vol) Nonidet P-40, 0.5% (wt./vol) sodium deoxycholate, RIPA buffer (10 mM Tris (pH 8), 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% (wt./vol) sodium dodecyl sulfate (SDS), 4 µg/mL aprotinin). Commercially available samples of murine vessel tissue for positive controls (PC) were previously ground and lysed in PMSF, then added to RIPA. The concentration of protein was measured according to the Bradford method (Kruger, 1994). SDS polyacrylamide gel (PAGE) was laden with 25 μg of protein and subsequently shifted to Amersham Hybond ECL Nitrocellulose Membrane (GE, Buckinghamshire, UK). Membrane block was performed in 0.03% Tween-20 in PBS and 5% (wt./vol) skim milk, followed by incubation with the monoclonal murine anti-alpha-smooth-muscle-actin (α-SMA) (Chemicon, Millipore, Schwalbach, Germany) as the primary antibody. The donkey-anti-mouse (excitation/emission maxima: 679/696 nm) and donkey-anti-mouse (excitation/emission maxima: 778/795 nm) (LiCor, Bad Homburg, Germany) were used as secondary antibodies. Blot analysis was performed using an infrared fluorescence detector (Odyssey Infrared Scanner, LiCor, Bad Homburg, Germany).

Preparation of total protein was perfomed using the Qproteome™ Mammalian Protein Prep Kit (Qiagen) according to the manufacturer’s protocols. Determination of protein concentration was performed using the DC Protein Assay (Bio-Rad) according to the manufacturer’s protocols. 15 μg of total protein extracts per lane were loaded onto 8% polyacrylamide gels. Proteins were transferred onto Amersham™ Hybond™ ECL Nitrocellulose Membrane (GE Healthcare) by Semi-Dry blot. The membrane was blocked in 5% (m/v) nonfat dried milk powder in Tris buffered saline with Tween (TBST). Then the primary antibodies diluted in 5% (m/v) nonfat dried milk powder in TBST (mouse anti-human EGFR clone H11 (Dako), mouse anti-β-Actin clone AC-15 (Sigma-Aldrich)) or 5% BSA in TBST (polyclonal rabbit anti-HER2/ErbB2 (Cell Signaling)) were incubated. After HRP conjugated secondary antibody (Dianova) incubation, the membrane was incubated with ECL reagents (Thermo Scientific). Chemiluminescence detection and imaging was done with the Fusion Fx7 System and the FusionCapt Advance Software (Vilber Lourmat).

All reagents and solvents were from Sigma-Aldrich unless otherwise indicated. All standard Fmoc-protected amino acids were supplied by Activotec. The Fmoc-protected N-alkyl glycine derivatives Fmoc-NPhe-OH and Fmoc-NLys-(N-ε-Boc)-OH were obtained from Polypeptide Labs. Biotin-PEG NovaTag resin was supplied by Merck Millipore. ESI-MS analysis was carried out by ASEPT (Queen’s University Belfast).
Human plasmin and thrombin were supplied by R&D Systems. Human neutrophil elastase (NE) and chymotrypsin were supplied by Calbiochem. Levels of active NE in cystic fibrosis (CF) sol were determined using the ProteaseTag^® Active Neutrophil Elastase Immunoassay, obtained from ProAxsis Ltd. The assay was performed exactly according to the manufacturer’s instructions.
SDS-PAGE was carried out using NuPAGE Novex 4–12% Bis-Tris protein gels (1.0 mm) using a PowerEase 500 power supply with a SeeBlue Plus2 pre-stained Protein Standard as a reference ladder, all supplied by Invitrogen. Western blotting was achieved with an X-Cell II Blot Module (Invitrogen) unto an Amersham Hybond ECL Nitrocellulose membrane (GE Healthcare). Luminata Forte Western HRP substrate was supplied by EMD Millipore. Streptavidin-HRP was supplied by Vector Laboratories.

Cell were harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8.0, and 1× PIC; Sigma-Aldrich, St. Louis, MO, USA) and incubated on ice for 1 h, followed by centrifugation (6000 rpm at 4 °C for 10 min). The supernatant lysate was supplemented with 6× SDS loading buffer containing 5% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and boiled for 10 min. The samples were separated by SDS-PAGE and transferred to Amersham Hybond ECL-nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). The following first antibodies were used: anti-H3 (Abcam, ab1791, Cambridge, UK) in 1:3000 dilution and anti-GFP (Abcam, ab6556, Cambridge, UK) in 1:1000 dilution. For chemiluminescent detection, secondary antibodies were applied: RAM-HRP (Dako, P0260, Santa Clara, CA, USA) and GAR-HRP (Dako, P0448, Santa Clara, CA, USA), followed by incubation with Immobilon Western Chemiluminescent HRP substrate (Merck Millipore, Burlington, MA, USA) and scanning using the Li-Cor 3600 C-DiGit Blot Scanner platform (Li-Cor, Lincoln, NE, USA).