Results: 5

1.
Figure 2

Figure 2. From: Overexpressing Human Membrane Proteins in Stably Transfected and Clonal Human Embryonic Kidney 293S Cells.

Workflow for the overexpression of mammalian membrane proteins in stably transfected and clonal HEK293S GnTI- cells.

Sarika Chaudhary, et al. Nat Protoc. 2012 February 9;7(3):453-466.
2.
Figure 4

Figure 4. From: Overexpressing Human Membrane Proteins in Stably Transfected and Clonal Human Embryonic Kidney 293S Cells.

Expression levels from stably transfected and clonal HEK293S GnTI- cell lines as determined by western blotting. Each lane corresponds to the “after spin” sample from a separate clonal cell line (clones 1 to 9), visualized via anti-FLAG western blotting. All clones were solubilized using DDM. See text for experimental details.

Sarika Chaudhary, et al. Nat Protoc. 2012 February 9;7(3):453-466.
3.
Figure 3

Figure 3. From: Overexpressing Human Membrane Proteins in Stably Transfected and Clonal Human Embryonic Kidney 293S Cells.

Dilution of tranfected HEK293S GnTI- cells prior to drug selection. Cells from one well of a 6 well tissue culture plate (2 mL total) are diluted into five 10 cm2 tissue culture plates, by plating the appropriate amount of cells (indicated in the boxes). Each 10 cm2 plate possess 10 mL of DMEM. The final dilution factor is indicated for each plate.

Sarika Chaudhary, et al. Nat Protoc. 2012 February 9;7(3):453-466.
4.
Figure 1

Figure 1. From: Overexpressing Human Membrane Proteins in Stably Transfected and Clonal Human Embryonic Kidney 293S Cells.

Expression testing of full-length human Rh membrane proteins in transiently transfected HEK293S GnTI- cells. 10 μg of RhAG, RhBG and RhCG, subcloned into pACMV-tetO, were separately transfected into HEK293S GnTI- cells grown in 10 cm2 tissue culture plates. Cells were induced with doxycycline (24 hours post transfection), harvested (24 hours post induction), and analyzed via anti-His Western blotting, as described in the text. Based on these results, RhAG and RhCG were chosen as candidates for stable cell line generation.

Sarika Chaudhary, et al. Nat Protoc. 2012 February 9;7(3):453-466.
5.
Figure 5

Figure 5. From: Overexpressing Human Membrane Proteins in Stably Transfected and Clonal Human Embryonic Kidney 293S Cells.

Medium and large scale HEK293S GnTI- cell cultures. As shown in panel A, up to 7 × 1 L spinner flasks can be maintained in a single CO2 incubator. Alternatively, up to 3 × 3 L spinner flasks can maintained (not shown). In panel B, 2 × 10 L WAVE bioreactors, using cellbags manufactured by GE Healthcare (left) and Flex Concepts Inc. (right), are shown. Relevant features of the cellbag and WAVE bioreactor, as discussed in the text, are highlighted.

Sarika Chaudhary, et al. Nat Protoc. 2012 February 9;7(3):453-466.

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