Anti mouse igg hrp linked whole ab

Protein extracts (200 μg) or recovered proteins from FPLC fractions (15 μl) were loaded onto 8% SDS-PAGE gel and run at 125 V for 80 min. The proteins were transferred to PVDF membranes (Thermo Fisher Scientific) by the wet-transfer method with transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, and 0.005% SDS) at 20 mA for 60 min at 4°C. The membranes were blocked with 5% skim milk in 1× PBST buffer (1× PBS containing 0.05% Tween 20). The following primary antibodies were used for immunoblotting: anti-V5 tag (mouse, R960-25, Invitrogen), anti-FLAG (rabbit, F7425, Sigma-Aldrich), anti-c-Myc (9E10) (mouse, 13–2500, Invitrogen), anti-ß-actin (mouse, ab8224, Abcam), and anti-histone H3 C-terminus (rabbit, ab1791, Abcam), all diluted 1:7,500 in 1× PBST buffer, and anti-Chp2 (rabbit [10 (link)]) diluted 1:1,000 in 1× TBST buffer (1× TBS containing 0.05% Tween 20). As secondary antibodies, anti-mouse IgG HRP-linked whole Ab (GE Healthcare) and anti-rabbit IgG, HRP-linked whole Ab (GE Healthcare), diluted 1:7,500 in 1× PBST buffer, were used. Membranes were incubated with Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore), exposed from 5 sec to 10 min with a ChemiDoc^™ MP Imaging System (Bio-Rad), and analyzed with Image Lab^™ software (Bio-Rad).

Escherichia coli cells from 2 ml culture were resuspended in 1 ml of chilled lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 5 mM imidazole, 0.07% mercaptoethanol, 0.1% Triton-X-100, 1× complete EDTA-free (Roche) and 1 mM PMSF) and the soluble fraction was isolated after sonication and centrifugation; 7.5 μl of the soluble protein lysate was loaded on SDS-PAGE gels for silver staining (Source Data for Supplementary Fig. 7). For the western blot analysis, 150 μl of the soluble protein lysate was precipitated with 400 μl acetone. After centrifugation at 4 °C for 30 min at 15,000 rpm, the pellet was extracted with 15 μl of 1× loading buffer (50 mM Tris-HCl (pH6.8), 2% SDS, 0.2% bromophenol blue, 100 mM DTT, 10% glycerol) and loaded onto an SDS-PAGE gel. Expression of recombinant proteins was assayed by western blot analysis with an anti His-Tag antibody (PGI proteintech Group; AB_11232599) at 1:20,000 dilution followed by incubation with Anti-Mouse IgG, HRP-Linked Whole Ab (GE Healthcare; AB_772209) at 1:50,000 dillution. Signals were detected with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and visualized with an ImageQuant LAS4000 (GE Healthcare). The signal intensities of the western blot analysis were analysed using ImageQuant TL v.8.1 (GE healthcare).

The indicated cells were transfected with the indicated plasmids and cultured for 2 days in 0.1% FBS-containing medium under normoxic conditions. Both cell lysates harvested with CelLytic M (MilliporeSigma) and culture medium were subjected to Western blotting using anti-myc epitope tag mouse monoclonal antibody (1000-fold dilution; Cell Signaling Technology, clone 9B11, catalog 2276) for the detection of exogenously expressed SPINK1 and its derivatives and anti-human β-actin mouse monoclonal antibody (200-fold dilution; Santa Cruz, clone AC-15, catalog Sc-69879) as primary antibodies, anti-mouse IgG HRP-linked whole Ab (5000-fold dilution; GE Healthcare Bioscience) as secondary antibody, and ECL Prime Western Blotting Detection Reagents (GE Healthcare Bioscience) for detection. The culture media were 17 times concentrated using Amicon Ultra-0.5 mL Centrifugal Filters, Ultracel-3K (Merck Millipore) before Western blotting according to the manufacturer’s instructions.