Generation of polyclonal antibody against fusion protein

189-bp Vsig1 cDNA fragment was amplified by RT- PCR using FuProF2 and FuProR2 primers. Then the PCR product subcloned into pGEMT-easy vector and transformed into DH5α cell (Invitrogen). The Hind3/BamH1 cDNA fragment was isolated from the pGEMT-easy vector and cloned into pET41a vector and the resulted construct was transformed into DH5α cell. The plasmid DNA of pET41-Vsig1 construct transformed into competent cell BL21DE3 (Novagen). Transformed cells were cultivated on LB-kanamycin plate at 37°C for overnight. 8 individual clones were cultivated in 5ml of LB+kanamycin medium at 37°C with shaking for overnight.

After 16 hrs, IPTG was added in each of cultivation up to 1mM concentration. After 3 hrs, cells were harvested by table centrifuge (at 5rpm, for 10 min) for analyse of fusion protein. Thus, the best clone giving high yield of fusion protein was selected for further large scale fusion protein purification.

2.2.13.2 Purification of fusion protein using GST affinity column

1 liter of bacterial culture was prepared for purification of the fusion protein.

The cells were harvested by centrifugation at 4000 X g for 20 minutes. And supernatants were discarded. The cells were resuspended in 50 ml of ice cold resuspension buffer (from the kit). The resuspended cell sample was sonicated for disruption of bacterial cells. Then the cell lysate was centrifuged at 39,000 × g for 20 min to remove the cell debris. The supernatant was followed into new falcon tube and filtrated though a 0.45 micron membrane to prevent clogging of the resin (syringe-end filters are convenient for this purpose). GST-Bind Resin was gently mixed by inversion until completely suspended. Using a wide-mouth pipette, 4 ml of GST-Bind-Resin was transferred to the small polypropylene columns (Novagen) and allowed to settle down by gravitation force. The prepared affinity column was washed with 5 volumes of GST Bind/Wash Buffer for calibration. The GST-Bind/Wash Buffer was allowed to drain to the top of the column bed and the prepared fusion protein extract was loaded through the column. The loading of fusion protein extract through the column was repeated three times for efficient binding. The final flow-through fraction was collected on ice and stored in -20C for further analysis. The column was washed with 20 ml of 1X GST Bind/Wash Buffer. At the end, the affinity bounded proteins were eluted with 7 ml of 1X GST Elution Buffer.

2.2.13.3 Immunization of rabbit

For immunization, two rabbits were injected with a denatured and native GST-VSIG1 fusion protein samples, respectively. Denatured GST-GST-VSIG1 fusion proteins were prepared by mixing with 1% SDS, 5mM DTT and boiled for 10 minutes. 500 mg (total volume 500 ml) of denatured and native fusion proteins were mixed 500 ml of Freund’s complete adjuvant, respectively (final total volume 1 ml). Before the first injection of the rabbits, approximately 10 ml of blood was bled from each rabbit for zero-control. Next boost injections were performed in 21 days interval with 500 mg

of denatured and native fusion proteins mixed with Freund’s incomplete adjuvant.

After 21 days of 3-rd boost injections, the rabbits were sacrificed and the entire blood was collected. The serum was isolated from blood sample by centrifugation at 2000g for 10 minutes at 4°C.

2.2.13.4 Purification of whole IgG from immunized rabbit serum

The whole IgG was purified using MAbTrap antibody purification kit (Amersham). The serum sample was centrifuged at 10 000 x g for 10 minutes to remove cells and debris. The supernatant was filtrated through a 0.45 µm filter.

Before application, the HiTrap Protein G-HP column and buffers were warmed to 37C. The column was equilibrated with 5 ml distilled water, and followed by 3 ml diluted binding buffer. Serum sample was applied through the column by syringe in slowly pumping. After applying of serum, the column was washed with 5–10 ml diluted binding buffer until no material appears in the eluent. After washing, the column was eluted with 3–5 ml diluted elution buffer. The eluted fractions were collected into 1.5 ml tubes containing neutralization buffer and stored at -20C.

(BioRad) active ester agarose was used. Affi-Gel 10 is N-hydroxysuccinimide ester crosslinked to agarose gel beads which is capable to bind covalently with ligands containing free alkyl or amino groups. 3 ml (1mg/ml) of GST-VSIG1 fusion protein was dialyzed against 5 liter of MOPS buffer (100mM MOPS, pH 7.5) for 24 hrs at 4°C to remove Tris derived from elution buffer. 3 ml of Affi-Gel 10 was washed three times with 9 ml of cold dionized water. Dialyzed 4 ml fusion protein was added to the washed Affi-Gel 10 pellet and the sample was incubated by rotating at 4°C for 4 hours for coupling. After coupling, 100µl of 1M ethanolamine-HCL (pH 8.0) was added to the coupling samples and incubated for further 1 hr for blocking. After blocking, the sample was centrifuged at 1000g for 5 min and the supernatant was removed for analyzing of binding efficiency (by Goomassie staining) comparing to non coupled

fusion protein sample. The pellet was washed 2 times with MOPS buffer and settled into the column.

2.2.13.5.2 Affinity purification of VSIG1 specific antibody

The prepared affinity column was washed with 10 ml of PBS two times.

Approximately 3 ml of anti-serum was applied to the column and followed by slowly dropping. The follow through was collected and the running was repeated at least 5 times. After last running, the column was washed with 10 ml PBS two times to remove residual serum materials. The column was eluted with 10 ml of elution buffer (100 mM of glycine-HCL pH 2.4, 150mM NaCl. 1 ml of elution fractions were collected separately and neutralized directly by adding 200 µl of 1M Tris-HCL (pH 8.0). The elution fractions from 2-5 contain highest concentration of affinity purified antibody.