Goat anti rabbit igg hrp conjugate

The peptide APTKAKRRVVQREKR corresponding to the epitope-tag on the B41-D7324 trimers was obtained from GenScript, as was a scrambled control peptide, KKQARRARPVREVKT). The peptides were used to coat Maxi-Sorp (Nunc) ELISA plate wells at 0.6 μg/ml. After standard washing and blocking procedures, rabbit sera were added at a dilution of 1/20 in PBS with 2% milk and bound antibodies were detected with a goat anti-rabbit IgG-HRP conjugate diluted 1/3000 (BioRad). As positive controls, sheep Ab D7324 was titrated from 10 to 0.013 μg/ml, and a pool of IgG purified from HIV-1 infected humans (HIVIG; supplied by the NIH AIDS Reagents Program, as #3957, lot # 140406–23) was titrated from 1000 to 1.3 μg/ml. The detection antibodies were rabbit anti-sheep IgG-HRP (Thermo) and goat anti-human IgG-HRP (BioRad), respectively.

Detection of GNPDA2 protein expression from transfected ADMSCs after stimulating differentiation to adipocytes for 10 days was carried out by western blot. The procedures were performed according to standard protocols or the manufacturers' instructions. Membranes were blotted with GNPDA2 polyclonal rabbit antibody (1 : 1000 dilution; 17105-1-AP, Proteintech), followed by goat anti-rabbit IgG HRP conjugate (1 : 5000; Bio-Rad Laboratories). Bands were visualized using the ECL Western Blot Kit (CWBIO, Beijing, China).

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).

Proteins were transferred to 0.45 μm Immobilon-P PVDF membrane (Millipore, Burlington, MA) in transfer buffer (25 mM Tris, 200 mM Glycine, 20% methanol) for 75 min at 500 mA at 4°C. The membranes were blocked with blotto (2.5% nonfat dry milk, 0.5% BSA, 0.5% NP-40, in TBST) for 30 min at room temperature and then incubated with rabbit anti-ZLD (1:750) (Harrison et al. 2010 (link)), or anti-Tubulin (DM1A, 1:5,000) (Sigma, St. Louis, MO), overnight at 4°C. The secondary incubation was performed with goat antirabbit IgG-HRP conjugate (1:3,000) (Bio-Rad, Hercules, CA) or antimouse IgG-HRP conjugate (1:3,000) (Bio-Rad) for 1 h at room temperature. Blots were treated with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) and visualized using the Azure Biosystems c600 or Kodak/Carestream BioMax Film (VWR, Radnor, PA).