Biology Reference
In-Depth Information
2.
Washing buffer; 100 m
M
Tris/HCl, 1 m
M
EDTA (pH 8.0). Elution buffer; wash-
ing buffer with 2.5 m
M
desthiobiotin (Sigma). Regeneration buffer; washing
buffer with 1 m
M
HABA (4-hydroxyazobenzene-2-carboxylic acid) (Sigma).
The above buffers are filtered through a 0.2-
m sterile filter prior to use.
3. StrepTactin (IBA, Göttingen) may be bought as free StrepTactin Sepharose
or as a ready-to-use column (1 mL column volume) and is claimed to bind up to
350 nmol of biotin per mL gel matrix.
µ
3. Methods
3.1. Transfection of 293-EBNA cells
1.
293 EBNA cells (Invitrogen, San Diego, CA) are cultured in growth media on
10-cm culture dishes at 37
°
C and in 5% CO
2
until 80% confluent.
2.
The cells are trypsinized and dispersed by trituration. They are then pelleted at
150
g
for 3 min, resuspended in 5 mL of growth media, and counted (a 10-cm dish
gives about 2
×
10
7
cells).
10
5
per mL in growth media and 800
3.
Cells are diluted to 6
×
µ
L of cells are mixed
with 10
g of purified circular plasmid DNA, (Qiagen or equivalent) and left for
5 min at room temperature. A control electroporation with no DNA added to the
cells is also carried out.
µ
4.
The cells are placed in a 4-mm electrode gap cuvette (Bio-Rad, Richmond, CA)
and electroporated with the following settings:
Capacitance
500
µ
F
Voltage
230 V
5.
After electroporation, the cells are left for 5 min to recover and then plated out on
a 10 cm culture dish in growth medium.
6.
Medium is changed daily and puromycin selection is initiated 48 h after transfec-
tion. After 6 d of selection, the negative control cells are dead, whereas the test
plate is 60-70% confluent with obvious areas of clonal growth having been
observed earlier.
7.
The plate is trypsinized and the cells split on to two 10-cm dishes, which are
allowed to grow for a further 2 d before the cells of one plate are cryopreserved in
liquid nitrogen.
8.
The other plate is allowed to grow for another 2 d until highly confluent. There
should be no gaps between cells and the cell sheet should appear thick and undu-
lating. As most of the contaminating proteins tend to be serum-derived, washing
to remove the selection media should be thorough. The plates should be drained
by being left at an angle for a minute to remove all residual medium. They are
washed twice with PBS with care taken to remove all the PBS. Eight mL of
serum free medium is added to the plate. The serum free media is replaced after
2, 4, and 6 d and the harvested media cleaned of debris by centrifugation and
stored at -20
°
C until analyzed by SDS-PAGE and/or immunoblotting.
3.2. Purification of Expressed Recombinant Protein
If the protein is expressed, cells can be thawed and the culture scaled up.
Generally, we grow the cells at this stage upon 5 to 20 dishes of 15-cm diam-
Search WWH ::
Custom Search