harpreet. k. dhiman 05/22/06. garp-2 expression in hek293s cells a total of 50 plates used. 1.25* 10...
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![Page 1: Harpreet. K. Dhiman 05/22/06. Garp-2 expression in HEK293S cells A total of 50 plates used. 1.25* 10 9 cells were used to express Garp-2 protein. Already](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697c00e1a28abf838cc9eff/html5/thumbnails/1.jpg)
Harpreet. K. Dhiman 05/22/06
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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.
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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.
![Page 4: Harpreet. K. Dhiman 05/22/06. Garp-2 expression in HEK293S cells A total of 50 plates used. 1.25* 10 9 cells were used to express Garp-2 protein. Already](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697c00e1a28abf838cc9eff/html5/thumbnails/4.jpg)
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.
![Page 5: Harpreet. K. Dhiman 05/22/06. Garp-2 expression in HEK293S cells A total of 50 plates used. 1.25* 10 9 cells were used to express Garp-2 protein. Already](https://reader036.vdocuments.us/reader036/viewer/2022062315/5697c00e1a28abf838cc9eff/html5/thumbnails/5.jpg)
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.
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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.
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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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ComparisonM
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