Pierce protein a igg purification kit

The anti-LPH polyclonal antibody was generated using female New Zealand white rabbits, as female is sensitive with lower doses of antigen and have higher response to immunization than male⁵⁶ . Female New Zealand white rabbits (6-8 weeks, 2 kg body weight) were immunized through repeated intradermal injections of LPH (1:1 emulsified in Freund’s adjuvant). After the final boost, blood was collected and serum was prepared. The polyclonal antibody was purified using Pierce™ Protein A IgG Purification Kit (Thermo Fisher Scientific). Immunoblot was performed to detect the presence of LPH in the culture supernatant of L. casei, L. rhamnosus, and L. paracasei. Probiotics were grown in Man, Rogosa, and Sharpe medium at 37 °C, 24 h without agitation. The cell lysates and culture supernatants were harvested, separated on SDS-polyacrylamide gel, and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5 % skim milk and incubated with rabbit polyclonal LPH antibody (1:5000) overnight. Expression of LPH was detected using goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:5000, Proteintech, catalog number SA00001-2) and enhanced chemiluminescence reagent kit. The uncropped scans of immunoblots were provided in the Source Data file.

C-LSLQRTGpSGG, C-LSLQRpTGSGG, and C-LSLQRTGSGG peptides were synthetized and purified in Molecular Biotechnology Core of Cleveland Clinic. Peptides were conjugation to KLH with glutaraldehyde. Four mice were injected into the peritoneum with an emulsion containing the antigen and an equal volume of Freund’s adjuvant. Complete Freund’s adjuvant was used for the first injection, Incomplete Freund’s was used for subsequent boosts, and no adjuvant was used for the final injection. Mice were injected at 3-wk intervals. 10 d after the third injection, the concentration of antibodies specific to each antigen was measured in the serum by ELISA. Spleen cells from the highest titer mouse were fused with SP2/0 cells by a standard PEG/DMSO method, and fused cells were selected using HAT media. 14 d later, supernatants were assayed by ELISA on the specific antigens. Cells from positive wells were expanded for cloning and individual cells producing antibodies were isolated from the mixed culture by the limiting dilution. Antibodies were developed in the Hybridoma Core of Cleveland Clinic. Antibodies were purified from tissue culture supernatants using Pierce Protein A IgG Purification Kit from Thermo Fisher Scientific.

Primary and secondary antibodies, and other reagents used for staining in this study are listed in Table 2. From the novel SYNPO2a-CT antiserum, the IgG fraction was purified from the complete serum using the Pierce ProteinA IgG purification kit (ThermoFisher Scientific) according to the instructions of the manufacturer.