Download MembranePro Functional Protein Expression System

Troubleshooting
Generating the
pEF6 Expression
Construct
For troubleshooting the problems you may encounter while generating your pEF6
expression construct containing your gene of interest , refer to the pEF6/V5-His
TOPO® TA Vector Kit manual (part no. 25-0279).
Transfection
The table below lists some potential problems and solutions that help you
troubleshoot the transfection step in your membrane protein expression
experiments.
Symptom
No VLPs recovered
and the control
transfection did not
work
Cause
293FT cells are
unhealthy
Solution
Ensure that the cells are healthy and greater than 90%
viable before transfection.
Use cells that have been subcultured for less than
16 passages.
Use cells that have been passaged 3–4 times after the
most recent thaw.
Density of 293FT culture
is too high
Do not allow cells to grow to confluency.
Transfection
unsuccessful
Follow the transfection protocol exactly as described
on pages 12–15. During transfection, pay particular
attention to the following points:
Ensure that the monolayer is 90% confluent at time of
transfection to avoid cytotoxicity and detachment of
cells during manipulation.
• Do not include antibiotics in the medium.
• Use Opti-MEM® I Reduced Serum Medium to
dilute transfection complexes.
• Because 293FT cells are easily dislodged, do not
squirt transfection complexes directly onto cell
monolayer.
Used cells other than
293FT
If you are using a cell line other than 293FT (i.e., a
stable cell line), we recommend testing your cell line
with the pEF6/V5-His-TOPO®/lacZ control (or a
similar reporter construct) to determine its
transfectability with Lipofectamine® 2000 before
attempting VLP formation.
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