Biomedical Engineering Reference
In-Depth Information
6. Caution: PFA is toxic. To prepare 3.7 % PFA, dissolve 1.48 g
of PFA in 30 ml sterile water by adding four drops of 10 N
KOH and heat the mixture at 50 °C in a hood for 15 min;
vortex gently a few times. Add 8 ml 1 M HEPES pH 7.4, mix
well, and fi lter the solution through a 0.22-
m membrane.
3.7 % PFA can be prepared fresh before immunofl uorescence
staining experiments; it can also be stored at −20 °C.
7. To prepare Mowiol mounting medium, add 2.4 g Mowiol
4-88 to 6 g of glycerol, and mix by stirring thoroughly, fol-
lowed by the addition of 6 ml distilled water. Incubate the
mixture at room temperature for 2 h. Add 12 ml of 0.2 M Tris
pH 8.5 and incubate at ~53 °C until the Mowiol has dissolved.
Clarify by centrifugation at 5,000 ×
g
for 20 min. Aliquot the
supernatant into vials and store at −20 °C.
8. The FreeStyle™ 293-F cells are mammalian cells derived from
the HEK293 cell line that is adapted to growth in suspension,
with a doubling time of about 24 h depending on the age and
handling of the cells. Cells should be split to 0.2-0.5 × 10
6
cells/
ml when the density reaches 1-3 × 10
6
cells/ml. Cell viability
and density can be assessed by trypan blue staining and hemo-
cytometer counting.
9. The culture volume should always be about 20 % of the fl ask
volume. The performance of the cells may be affected if the
culture volume is 30 % or more of the fl ask volume.
10. The amount of DNA required for transfection depends on the
volume of culture, the expression construct, and the size of the
protein. A strong promoter or a smaller protein usually requires
less DNA and shorter transfection time to reach a similar level
of protein production. A recommended starting condition is
1
ʼ
g DNA/ml of culture; further optimization may be
required. In addition to PEI, 293fectin™ reagent (Invitrogen)
can be used for transfecting FreeStyle™ 293-F cells. In our
hands, 293fectin™ reagent gives higher protein production for
secreted proteins when compared with the use of PEI, but the
two reagents appear to perform almost equally well for cyto-
solic proteins. Please refer to the manufacturer's protocol for
details regarding the 293fectin™ reagent.
11. Time required for optimal expression varies between proteins.
If the protein is expressed in FreeStyle™ 293-F cells for the
fi rst time, harvest cells at multiple time points to determine the
shortest time required to get the highest level of protein and
whether the protein is intact or degraded.
12. High-salt wash can help reduce nonspecifi c, ionic binding.
13. The elution conditions vary between proteins. Please refer to
Note 5
for the choices of FLAG peptides. A total of fi ve column
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