Guinea pig serum EU145 was raised against the N-terminal 314 residues of the predicted 2.2 MDa isoform of Ce-titin. Antigens was expressed as GST fusion proteins in E. coli. cDNAs encoding antigenic polypeptides were amplified by RT-PCR using Pfu polymerase (Stratagene) and primers containing added XhoI and EcoRI sites. The 5' and 3' primers for EU145 were 5'-GTACGAATTCATGGAGGGCAACGAGAAGAAAGG-3' and 5'-GATGCTCGAGCATTGGGTCAAAGGCAGTGGTCG-3', respectively. Amplified product was cloned into pGEX-6P-1 (Amersham Pharmacia Biotech).T he GST fusion proteins were expressed and purified. Milligram quantities of fusion proteins were supplied to Spring Valley Laboratories (Sykesville, MD) for antiserum production.
ttn-1