ELISA

Somatotype Attitudinal Mean (SAM)

For construction of desired clones, 1-2 μg of DNA was used for restriction digestion. In a typical reaction, 2-5units of restrictionenzymeswereused in the total reaction volume of 50μl along with5μl ofrecommended buffer (supplied as 10X digestion buffer). The reaction mixture was incubated for 3-5 hat 37oC. The digested DNAwasthenloaded along with DNA size marker and separated on agarose gel electrophoresis. The gel was visualized over a UV illuminator and section containing the desired DNA fragment was carefully sliced out. The sliced agarose gel was then processed using commercially available Gel Extraction kit (Qiagens) for this purpose

Digestion and elution of DNA

On the HPLC system (Waters Empower Software), the instrument method was set to the programme ‘tritium small coloumn’ and the method set was set to ‘tritium small column’. The ‘set up’ icon was selected and once the flow rate was 1 mL/min, the ‘prepare’ icon was selected. A gradient was generated by mixing buffer A and bufferB as described below (Section 2.1.6.6). The injector was moved to the load position and sample was injected using a 1 mL syringe. The injector was moved to the inject position and the ‘inject’ icon was pressed immediately. On the fraction collector, the ‘run’ button was pressed immediately. The syringe was rinsed 5 times with buffer A

HPLC set upA strong anion exchange partisphere SAX 4.6 mm diameter and 125 mm length column (Whatman) was fixed to the HPLC system (Waters 515 pumps). The column was equilibrated with buffer A (1 mM EDTA) (Section 2.1.6.6) overnight at 100 μL/min flow rate.Before starting the HPLC run, the fraction collector (BioRad 2110) was prepared by placing the outlet tube to vial 1 on the fraction collector. Fresh FACS tubes were placed at fraction numbers 40-65 on the fraction collector. The flow rate on the fractioncollector was set to 1 mL/min and it was kept ready.The SAX column was allowed to equilibrate (warmup programme) with Buffer A by slowly increasing theflow rate from 0.1 to 1 mL/min over a period of 70 min

Purification of radiolabelled IP7by HPLC method