Harpreet. K. Dhiman 05/22/06

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 (2g/ml) and sodium Butyrate (5mM) followed by harvesting of cells after 2 days.

•Purification using strep-Tactin sepharose gravity flow column.

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.

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.

Western blot of Garp-2 expression from three systems using equivalent amount of cells.

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." Nature 400(6746): 761-6.

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 10l was loaded. In the next lane, 10l Garp-2 protein from Cos-1 cells, followed by 40l protein from HEK-293S cells was loaded directly without dilution.

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.

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." Nature 400(6746): 761-6.

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Coomassie stained PVDF membrane after transfer of Garp-2 protein from gel to membrane.

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Western blot of purified Garp-2 expression from three systems using equivalent amount of cells.

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Sterp-tactin HRP conjugate from IBA GmbH company was used for detection of sterp-tagged protein.

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Thanks