Anti mouse igg peroxidase antibody

LTA was extracted from over-night cultures normalized based on OD₆₀₀ readings. Samples were centrifugated and resuspend in 300 μl of PBS, lysed using 0.1mm zirconia/silica beads (Biospec Product) in MP Biomedicals Instrument FastPrep-24, 3X30sec at maximal speed. Pellets were resuspended in 100 μl of 2X SDS-page loading buffer, incubated at 95°C for 30 min, centrifuged at 17,000 x g for 5 min. 10 μl aliquots were separated on 15% SDS-PAGE gels and subsequently transferred to a PVDF membrane. LTA was detected using the monoclonal polyglycerolphosphate-specific LTA antibody (1:250; Clone 55 from Hycult Biotechnology) and the anti-mouse IgG-Peroxidase antibody (1:5000, Sigma) and blots were developed by enhanced chemiluminescence (ECL-prime Amersham-GE-healthcare).

The growth complementation assays were performed as previously described^{29 (link)}. In brief, His-tagged versions of KdpA, KdpFABC and KtrB, respectively, were expressed in E. coli LB2003, a strain lacking all endogenous K⁺ uptake systems. LB2003 transformed with empty vector pBAD18 served as negative control. Growth curves were recorded for 24 h at different K⁺ concentrations (1–115 mM referred to as K1–K115). At K10 and below the strain only grows sufficiently, if the expressed protein complements the lacking transport systems. Protein production was confirmed by Western blotting analysis of a K30 sample after 24 h using an anti-His antibody from mouse (dilution 1:3000, Sigma-Aldrich, cat.no. H1029) and secondary anti-mouse IgG-peroxidase antibody produced in goat (dilution 1:20,000, Sigma-Aldrich, cat.no. A2554).

The hemocyanin-specific IgM level was determined by an ELISA kit (Aquatic Diagnostic, Ltd.) as previously described (35 (link)). Briefly, PBS as blank or fish sera from naive, hemocyanin-immunized, or pre-immune animals as control diluted 1:100 were added to the hemocyanin precoated 96-well ELISA plates, followed by the monoclonal antibody anti-seabream IgM at 1:1,000. Then, an anti-mouse IgG peroxidase antibody produced in goat (Sigma-Aldrich) diluted 1:1,000 was included as a reporter. Finally, the chromogen TMB (Sigma-Aldrich) was added, and the plates were incubated at 22°C. The reaction was stopped with the addition of 50 μl/well of 2 M H₂SO₄, and the absorbance was read at 450 nm on a SPECTROstart nano (BGM; LabTechnologies). The plotted values resulted from subtracting the absorbance from the control wells.

S2R + cell were transfected with 300 ng of pEsCry1. Culture plates were placed under fluorescent white lighting (7.3 klux) at 25 °C for 8 hours. Dark control plates were wrapped in aluminium foil and incubated next to the light-treated plates. Proteins were: extracted in TritonX-100 lysis buffer (20 mM Hepes pH 7.5; 100 mM KCl; 2.5 mM EDTA pH 8.0; 5% glycerol; 0.5% TritonX-100; 1 mM DTT; 1X Complete Protease Inhibitor Cocktail-Roche); separated by SDS-PAGE in a NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher); and transferred to a nitrocellulose membrane (Bio-Rad) by routine methods using a wet blottingsystem (Thermo Fisher). Western Blot membranes were incubated with monoclonal anti-HA antibody (Sigma) and then Anti-Mouse IgG-Peroxidase antibody (Sigma). Chemioluminescence reaction was performed with fresh made ECL buffer. Protein bands were visualized using Amersham Hyperfilm ECL (GE Healthcare). Relative abundance of EsCRY1 was normalized to the total protein amount per lane evaluated by Ponceau S staining^{65 (link)} (full-length blot and Ponceau S staining are shown in Supplementary Figure S8D). For quantification of the immunodetected signals, each film was analysed with Image J software (http://rsb.info.nih.gov/ij).

Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (10mM Tris-HCl pH 8.0, 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). Lysates were clarified and subsequently separated by SDS-PAGE on 10% polyacrylamide and wet transferred to PVDF. The membrane was blocked with 5% milk solution in Tris-buffered saline Tween 20 (TBST) at room temperature, and primary antibody was added at a dilution of 1:1000 in milk buffer. Secondary antibody was added at a dilution of 1:10,000 in milk buffer. Following each antibody incubation, the membrane was triple rinsed with TBST. Chemiluminescent visualization of blots was carried out using visualization solution made up of two buffers (buffer 1: 2.5 mM luminol, 0.396 mM coumaric acid, and 100 mM Tris-HCl pH 8.5; buffer 2: 0.0192% hydrogen peroxide, 100 mM Tris-HCl pH 8.5) mixed immediately before visualization.
The following primary antibodies were used: GAPDH loading control antibody MA5-15738 (Thermo Fisher Scientific), anti-RIOK3 SAB1406721 (Sigma), and anti-TRA2-β antibody ab31353 (Abcam). HRP-conjugated secondary antibodies used were anti-Mouse IgG peroxidase antibody produced in goat A2554 (Sigma) and anti-Rabbit IgG peroxidase antibody produced in goat A0545 (Sigma).

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). Clarified lysates were separated by SDS-PAGE on 10% polyacrylamide and wet transferred to PVDF. The membrane was blocked with 2.5% dry milk solution in Tris-buffered saline Tween 20 (TBST) at room temperature. Primary antibodies used include: Monoclonal M2 anti-FLAG (MilliporeSigma, Burlington, VT, USA), GAPDH loading control antibody MA5-15738 (Thermo Fisher Scientific), anti-RIOK3 SAB1406721 (Sigma). Following primary antibody incubation, the membrane was triple rinsed in TBST. HRP- conjugated secondary antibody used was anti-Mouse IgG peroxidase antibody produced in goat A2554 (Sigma). Following secondary antibody incubation, the membrane was triple rinsed with TBST. Chemiluminescent visualization of blots was carried out using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and a Fujifilm LAS 3000 imager.