Login

Join for Free!
118895 members
table of contents table of contents

A new method for generating high and stable protein expressing cell lines …


Biology Articles » Biotechnology » Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection » Figures

Figures
- Efficient and reproducible generation of high-expressing, stable human cell lines without need for antibiotic selection

mcith_13-1.jpg Figure 1 Expression of E1 proteins. Western Blot analyses for E1A and 21-kDa E1B proteins expressed in 10 E1-transformed amniocyte cell pools. Cells were lysed and proteins were size fractionated on a SDS-containing polyacrylamide gel. Proteins were transferred to nitrocellulose and probed with anti-E1A or anti-E1B 21-kDa antibody. Protein lysates from HEK293 or primary amniocytes were used as control.

(Click image to enlarge)

mcith_13-2.jpg Figure 2 Expression of hAAT protein. Western Blot analyses for hAAT protein expressed in 10 E1-transformed amniocyte cell pools. (A) For detection of secreted hAAT, proteins in the cell culture medium were fractionated on a SDS-containing polycrylamide gel. (B) For detection of intracellular hAAT, cells were lysed and proteins were separated on a SDS-containing polyacrylamide gel. Intracellular and secreted hAAT was visualized using a monoclonal anti-hAAT antibody. For control proteins from untransformed amniocytes and hAAT purified from human plasma were used.

(Click image to enlarge)

mcith_13-3.jpg Figure 3 Quantitation of hAAT expression in 6 amniocyte cell pools. Cells in different passages were plated into 6-well plates, the supernatants were collected after 72 hours and the amounts of secreted hAAT were analysed by ELISA.

(Click image to enlarge)

mcith_13-4.jpg Figure 4 Quantitation of hAAT expression in 6 clonal amniocyte cell lines. Single cell cloning was performed from amniocyte cell pools Z171-5A and Z171-5B in passages 26 and 27, respectively. Clonal cells in different passages were plated into 6-well plates, the supernatants were collected after 48 hours and the amounts of secreted hAAT were analysed by ELISA.

(Click image to enlarge)

mcith_13-5.jpg Figure 5 Glycoanalyses of hAAT. Proteins from cell culture medium of amniocyte cell pool Z171-5A were treated with increasing amounts of (A) PNGaseF or with (B) Neuraminidase and fractionated on SDS-containing polyacrylamide gels. Upon transfer on nitrocellulose Western Blot analyses were performed using a monoclonal anti-hAAT antibody. For controls hAAT purified from human plasma was used. (C) A- and B-type oligosaccharide chains of hAAT and cut positions of PNGaseF and Neuraminidase.

(Click image to enlarge)

 


rating: 3.00 from 2 votes | updated on: 29 Nov 2008 | views: 12024 |

Rate article:







excellent!bad…