Goat anti mouse igg hrp conjugate

Expression analysis of proteins during in vitro cardiac differentiation was carried out by western blot technique. Whole cell lysates were isolated from differentiated samples by using 1x passive lysis buffer (5x Passive lysis buffer, Promega, cat. no. E1941). Concentration of the whole cell lysate was determined by the Bradford's method as mentioned above. The protein samples were stored in 6X Laemmli dye [59 (link)] and 20 µg of the quantified protein was then loaded in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE) using Bio-Rad Mini-Protean 3 cell, cat. no. 67S/11919. The protein was then transferred to polyvinylidene fluoride (transfer membrane, Immobilon-P, Millipore, cat. no. IPVH00010) and the membrane was hybed with anti-RYBP antibody (Anti-DEDAF, Merck Millipore, cat. no. AB3637, 1 : 1000) and anti-PLAGL1 antibody (Anti-Zac1 antibody (C-7), Santa Cruz, cat. no. sc-166944, 1 : 1000). Bio-Rad Goat-anti-mouse IgG-HRP conjugate, (cat. no. 172-101, 1 : 2000) and Merck Millipore Goat-anti-Rabbit IgG-HRP conjugate (cat. no. AP132P, 1 : 2000) were used as the secondary antibodies. The membranes were washed with TBST buffer (for five times with10 min of gentle shaking and then hybed with Immobilon Western, Chemiluminescent HRP Substrate, Millipore, cat. no. WBKLS0500. Alliance Q9 system (UVITECH) was used to capture the chemiluminescent signals.

Cell lysates were obtained using modified radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Complete, Boehringer Mannheim, Mannheim, Germany), NaF (50 mM), PMSF (1 mM), aprotinin, leupeptin, pepstatin (1 ng/ml) and orthovanadate (2 mM) (Sigma-Aldrich). The following primary antibodies were used for immunodetection: rabbit anti-total AKT, anti-phospho-AKT (Thr 308) and anti-phospho AKT (Ser 473) and used at 1:1000 dilution (cell signaling), rabbit anti-total Erk1/2 (dilution 1:2000) and rabbit anti-phospho-Erk1/2 (Thr202/Tyr204) (dilution 1:1000) (cell signaling), mouse anti β-actin used at 1:5000 dilution (Sigma-Aldrich) and rabbit anti-vinculin used at 1:30000 dilution (Abcam). Goat anti-rabbit IgG HRP-linked (cell signaling) and goat anti-mouse IgG HRP-conjugate (Bio-Rad) were used as secondary antibodies at 1:2000 dilution.

Kidney organoids were washed with PBS and lysed with 10mM Tris (pH 7.5) containing 1% sodium dodecyl sulfate (SDS) and 1 mM NaVO₄. Equal amounts of protein were separated on 7% SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (IB401001; Invitrogen, Carlsbad, CA, USA) using the iBlot2 Gel Transfer Device (IB21001; Invitrogen). Membranes were blocked with 5% skim milk in PBS with 0.1% Tween 20 detergent (T-PBS) for 1 h at room temperature and incubated with anti-NCC (ab224762, 1:500; abcam) and biotinylated Lotus Lectin (LTL, B-1323,1:100; Vector Laboratories, Burlingame, CA, USA), E-cadherin (ECAD, 610405, 1:500; BD Biosciences, San Jose, CA, USA), and b-actin (1:2000, 3700; Cell signaling Technology, Danvers, MA, USA) at 4 °C overnight. Membranes were then washed 3 times with T-PBS and incubated for 1 h with a goat anti-rabbit IgG-HRP conjugate (1706515, 1:1000; Bio-Rad, Hercules, CA, USA), a goat anti-mouse IgG-HRP conjugate (1706516, 1:1000; Bio-Rad), and a Strep Tactin-HRP conjugate (1610381, 1:1000; Bio-Rad). After 4 washes with T-PBS, the protein of interest was detected using an enhanced chemiluminescence system (WSE-7110, ATTO Corp., Tokyo, Japan). Quantification of relative densities was performed with the control group set at 100%; densities were normalized to those of b-actin bands from the same gel (Quantity One version 4.4.0; Bio-Rad).

Cells (1.5×10³) from each cell line were lysed in RIPA buffer (Cell Signaling Technology) with 0.5% SDS and complete protease inhibitors (Roche) by mechanical disruption and freeze thawing; the cell mixtures were then reduced and denatured in NuPage LDS Sample Buffer (Life Technologies) with 8% β-mercaptoethanol, at 95°C for 5 minutes. The samples were run next to the Precision Plus Duel Color Standards (Bio-Rad) in a 4–12% Bis-Tris polyacrylamide gel (Life Technologies). Expression of each reporter protein and self-cleavage of the viral 2A sequences were evaluated by Western blots with a DsRed rabbit polyclonal antibody (Clonetech) diluted 1∶3000, firefly luciferase mouse monoclonal antibody (Abcam) diluted 1∶3000, HSV-1 thymidine kinase goat polyclonal antibody (Santa Cruz Biotechnology) diluted 1∶250, and GAPDH rabbit polyclonal antibody (Sigma-Aldrich) diluted 1∶5000. Secondary antibodies used were a goat anti-rabbit IgG HRP conjugate diluted 1∶3000 (Cell Signaling Technologies), goat anti-mouse IgG HRP conjugate diluted 1∶3000 (Bio-Rad), and donkey anti-goat IgG HRP conjugate diluted 1∶2500 (Promega).

Ten different Salmonella antibodies were selected among commercially available antibodies and are listed in Table 1. The antibodies were produced in mouse or rabbit using diverse immunogens, including LPS (whole or partial), flagella, and whole cell preparation. Ab 3, Ab 5, Ab 6, and Ab 7 were rabbit polyclonal, whereas Ab 1, Ab 2, Ab 4, Ab 8, Ab 9, and Ab 10 were mouse monoclonal (Table 1). They were purchased from Abcam (USA.) and Invitrogen (Waltham, USA). The secondary antibodies used were goat anti-mouse IgG–HRP conjugate (#1706516; Bio-Rad Laboratories, Inc., USA), goat anti-rabbit IgG–HRP conjugate (#31460; Invitrogen), goat anti-mouse IgG–AP conjugate (A3562; Sigma-Aldrich, USA), and goat anti-rabbit IgG–alkaline phosphatase (A3687; Sigma-Aldrich).

Quantification of protein expression levels in cell lines was performed by immunoblotting, as previously described [12 (link)]. Primary antibodies used were: PIK3R3 (1:1000, Cell Signaling, #11889), PTEN (1:1000, Cell Signaling, #9552), pAkt (Ser473, 1:500, Cell Signaling, #4060L), Akt (1:1000, Cell Signaling, #9272), and tubulin (1:20000, Sigma, T5168). Secondary antibodies used were: goat anti-mouse IgG-HRP conjugate (1:5000, BioRad, #170–6516), goat anti-rabbit IgG-HRP conjugate (1:5000, BioRad, #170–6515).