Harpreet. K. Dhiman 05/22/06

1. Harpreet. K. Dhiman 05/22/06

2. Garp-2 expression in HEK293S cells • A total of 50 plates used. • 1.25* 109 cells were used to express Garp-2 protein. • Already screened stable cell line clone was expanded. • The cells were induced by using tetracycline (2g/ml) and sodium Butyrate (5mM) followed by harvesting of cells after 2 days. • Purification using strep-Tactin sepharose gravity flow column.

3. Garp-2 expression in Tetracycline inducible Cos-1 cells • A total of 50 plates used. • 1.25* 109 cells were used to express Garp-2 protein. • DEAE-Dextran method was used for transfection. • Cells were harvested after 3 days. • Purification using strep-Tactin sepharose gravity flow column.

4. Garp-2 expression in baculovirus expression system. • 1.25* 109 cells were used to express Garp-2 protein. • MOI 8 (multiplicity of infection)was used to infect the • sf-9 insect cells in exponential phase • Cells were harvested after 3 days. • Purification using strep-Tactin sepharose gravity flow column.

5. Western blot of Garp-2 expression from three systems using equivalent amount of cells. Figure 1: Western blot analysis of StrepTag-Garp-2 protein expression from Bac-Bac baculovirus expression system by sf-9 cells, transient transfection by Cos-1 cells and stable HEK293S cells. To detect the protein an antibody against a peptide from c-terminus of Garp-2 (290-299) was used. In the first sf-9 cells lane, purified recombinant Garp-2 was diluted 1:100 and 10l was loaded. In the next lane, 10l Garp-2 protein from Cos-1 cells, followed by 40l protein from HEK-293S cells was loaded directly without dilution. Garp-2 antibody was made against a peptide from c-terminus of Garp-2 (290-299). From: Korschen, H. G., M. Beyermann, et al. (1999). "Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors." Nature400(6746): 761-6.

6. Stains-All gel of Garp-2 expression from three systems using equivalent amount of cells. Figure 1: The Stains-All gel of purified recombinant Garp-2 protein HEK293S, Cos-1 and sf-9 cells. In the first and second lane, 40µl of purified recombinant Garp-2 from HEK293S and Cos-1 cells was loaded followed by 5 µl Grap-2 from sf-9 cell.

7. M Sf-9 ( 1:10) 10ul HEK 40ul Cos-1 10ul Cos-1 5ul Sf-9 ( 1:50) 10ul Sf-9 ( 1:100) 30ul Sf-9 ( 1:50) 20ul Sf-9 ( 1:100) 20ul Sf-9 ( 1:100) 10ul Western blot of purified Garp-2 expression from three systems using equivalent amount of cells. Garp-2 antibody used for western blot was obtained from Germany. The antibody was made against a peptide from c-terminus of Garp-2 (290-299). Source:From: Korschen, H. G., M. Beyermann, et al. (1999). "Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors." Nature400(6746): 761-6.

8. M Sf-9 2ul Sf-9 1ul HEK 10ul Sf-9 4ul HEK 10ul M Cos-1 10ul Cos-1 10ul Coomassie stained PVDF membrane after transfer of Garp-2 protein from gel to membrane.

9. M Cos-1 10ul Cos-1 5ul Sf-9 ( 1:10) 10ul HEK 40ul Sf-9 ( 1:50) 10ul Sf-9 ( 1:100) 10ul Sf-9 ( 1:100) 20ul Sf-9 ( 1:100) 30ul Sf-9 ( 1:50) 20ul Western blot of purified Garp-2 expression from three systems using equivalent amount of cells. Sterp-tactin HRP conjugate from IBA GmbH company was used for detection of sterp-tagged protein.

10. M M Cos-1 10ul Cos-1 5ul Sf-9 ( 1:10) 10ul HEK 40ul Sf-9 ( 1:10) 10ul HEK 40ul Cos-1 10ul Cos-1 5ul Sf-9 ( 1:50) 10ul Sf-9 ( 1:100) 30ul Sf-9 ( 1:50) 20ul Sf-9 ( 1:100) 20ul Sf-9 ( 1:100) 10ul Sf-9 ( 1:50) 10ul Sf-9 ( 1:100) 10ul Sf-9 ( 1:100) 20ul Sf-9 ( 1:100) 30ul Sf-9 ( 1:50) 20ul Comparison

11. Thanks