Changes in the Temporal and Spatial Expression: MATERIALS AND METHODS(2)

7 Jun

Northern Blot Analysis

Samples of total RNA were dried under vacuum centrifugation, suspended in denaturing solution, and loaded onto 2.2 M formaldehyde-1% agarose gels. The RNA size markers (RNA ladder; Life Technologies, Gaithersburg, MD) were electrophoresed on the same gel to determine transcript size. The RNA was transferred by diffusion onto nylon membranes (Micron Separation, Inc., Westboro, MA) for 12 h, then the filter was removed and baked in a vacuum oven at 65°C for 2 h. Hybridization probes were prepared by random prime labeling the rat Hp58 cDNA previously isolated and cloned from the RNA fingerprinting analysis with [a- 32P]deoxy-CTP (3000 Ci/mmol; iCn Biomedicals, Costa Mesa, CA) according to the manufacturer’s protocol (Life Technologies). Hybridization conditions and blot washes were carried out as described in detail elsewhere.

Generation of H/358 Antibody

Computer analysis was used to search for potential antigenic regions in the mouse Hp58 protein. One region of hydrophilicity was identified from amino acids 280 to 285 (DEEDRR). Eight copies of the sequence (DEEDRRY-FKQQEILLWRK, Map-peptide) were synthesized, and the synthetic peptide was emulsified in Freund’s complete adjuvant. Rabbits were immunized by standard methods, and the Hp58 titer was determined from blood samples collected over a 10-wk period. An ELISA was developed using the Map peptide attached to plates as the solid phase (1 ^g/well), and a goat anti-rabbit IgG-horse radish peroxidase conjugate with a peroxidase dye as the detection system. The reciprocal of the serum dilution that resulted in an optical density at 492 nm of 0.2 was used to determine the titer. The sera from one rabbit provided a titer of 214 500 10 wk after the first injection. The preimmune serum and the Hp58 antiserum were collected from this rabbit and affinity purified by passage over protein A columns. Bound immunoglobulin proteins were eluted with acetic acid. The eluants were concentrated against polyethylene glycol 20 000. Total protein concentration in the preimmune serum (3.2 mg/ml) and the Hp58 antiserum (1.2 mg/ml) was determined using the BioRad reagent.