Rabbit Immunization and Antibody Purification

New Zealand white rabbits (Harlan Laboratories, Prattville, AL) were initially immunized with 100 µg of purified GST-TIP-1 protein premixed in a 1∶1 ratio by weight with Titermax adjuvant (CytRx Corporation, Los Angeles, CA). One month after the initial immunization, the animals were boosted with same amount of antigen twice with 2 weeks interval without the adjuvant. Blood samples were periodically taken for antibody titration and specificity analyses by ELISA or western blot. When the anti-TIP-1 antibody reached the designated high titer, the animals were sacrificed to collect the antiserum. The antiserum was purified by passage through protein A plus protein G columns (sigma) to purify IgGs. TIP-1 specific antibodies were prepared from the purified IgGs by tandem absorption with bacterial proteins and the purified GST protein-conjugated sepharose-4B (Sigma) to remove the IgGs that might bind proteins other than TIP-1. The final antibody was dialyzed against PBS and concentrated via Amicon centrifugal filters (Millipore, Billerica, MA). Specificity of the TIP-1 specific antibody was validated with whole cell staining and western blot analyses of whole cell lysates. All animal studies were conducted as approved (protocol ID: M/08/051 and M/08/592) by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University.