Changes in the Temporal and Spatial Expression: MATERIALS AND METHODS(3)

9 Jun


Antibody Specificity

To assess antibody specificity, the rat Hp58 cDNA was expressed as a fusion protein and reacted with the Hp58 antibody on Western blots. The Hp58 cDNA that had been isolated and cloned from the RNA fingerprinting analysis was amplified by PCR using Hp58 gene-specific primers. The resulting PCR product was subcloned in frame into the BamHI and £coRI sites of the pGex-2T expression vector (Pharmacia, Piscataway, NJ), and XL-1 bacteria were transformed with pGex-Hp58 and pGex vector alone. Positive colonies were isolated and the DNA was sequenced to verify the Hp58 open reading frame. Fresh overnight cultures of wild-type vector and vector containing the Hp58 cDNA were diluted 1:50 in Luria broth medium.

Expression of glutathione-S-transferase (GST) and GST-Hp58 fusion proteins were induced by addition of isopropyl-p-D-thiogalac-topyranoside (IPTG: 1 mM) for 2 h. Samples from noninduced and induced XL-1 bacterial cells were centrifuged for 2 min, and the bacterial pellet was suspended in sample buffer (10% glycerol, 4% SDS, 50 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.05% bromphenol blue, and 4% mercapto-ethanol). The samples were boiled for 2 min, and the su-pernatants were loaded on 10% SDS polyacrylamide gels. One of the gels was stained with Coomassie brilliant blue to visualize the proteins. Proteins on the other gel were transferred to a nitrocellulose membrane using methods standard in our laboratory. The blot was reacted with Hp58 antibody (50 ^g/ml), and immunoreactive proteins were visualized after treatment of the blot with the TMB substrate kit for horse radish peroxidase (Vector Laboratory, Burlingame, CA).