Biomedical Engineering Reference
In-Depth Information
Fig. 2
Immunofl uorescence micrographs of HeLa cells depleted of GCC185 by siRNA for 72 h followed by
transfection of GCC185 rescue plasmid.
Left column
, expression of the indicated rescue construct detected
using chicken anti-Myc and Alexa Fluor 555 goat anti-chicken antibodies.
Right column
, MPR localization
detected by 2G11 mouse anti-cation-independent MPR and Alexa Fluor 488 goat anti-mouse antibodies.
Approximate cell outlines are indicated
4
Notes
1. Glass fl asks may be used after thorough cleaning and autoclav-
ing to avoid potential contamination. Glass fl asks can be fi rst
soaked in dilute bleach (5-10 %) overnight. Rinse the fl asks
with water to remove bleach and any residual detergent. Fill
each fl ask with water up to 50 % of the total volume before the
fi rst autoclaving. Pour out the water and autoclave the fl asks
for a second time.
2. Filtering the PEI solution is both for sterility and to enhance the
effi ciency of transfection, as the presence of undissolved PEI can
interfere with transfection by precipitating DNA plasmids.
3. Thawed PEI can be kept at 4 °C and should be used within 2
weeks. Test the transfection effi ciency before using thawed PEI
that has been kept at 4 °C for more than 2 weeks.
4. Anti-FLAG M2 is a mouse monoclonal antibody that binds
FLAG-tag (or tandem FLAG) at the N-terminal, Met-N-
terminal, C-terminal, and internal locations of fusion proteins.
The calcium-dependent anti-FLAG M1 is an alternative, if
working with N-terminal (not Met-N-terminal) FLAG fusion
proteins. In addition to the use of FLAG peptide, proteins can
also be eluted with a buffer containing EDTA when using the
M1 resin.
5. Construct containing 3× FLAG fusion must be eluted with 3×
FLAG peptide. If the construct contains only 1× FLAG-tag
fusion, FLAG peptide can be used for elution.
