Biomedical Engineering Reference
In-Depth Information
3.9 Inhibition
of Unconventional
Secretion Using
Knockout Strains
1. Transform the knockout strains with the relevant plasmids (
see
Subheading
3.2
) (
see
Note 16
).
2. Pre-cultivate the transformants and wash with fresh medium
(
see
steps 1-6
of Subheading
3.3
).
3. Inoculate the cells in fresh 25 mL SD + HM medium to an
OD
600
of 0.3.
4. Incubate the culture medium at 25 °C with continuous shaking
for 4 h.
5. Filter the culture medium, concentrate, and lyophilize. Store
at −80 °C until further use (
see
steps 1-17
of Subheading
3.4
).
6. Resuspend the lyophilized sample in 25
ʼ
L of 1× buffer for
L for SDS-PAGE.
7. Perform SDS-PAGE and Western blotting (
see
Subheadings
3.6
and
3.7
).
8. Perform statistical analysis to evaluate secretion inhibition by
the knockout strains (
see
Note 17
).
SDS-PAGE and use 10
ʼ
4
Notes
1. The competent cells can be stored at −80 °C by freezing slowly.
2. Use transformants within 2 weeks after transformation. It is
recommended that freshly transformed cells be used for each
experiment.
3. It is recommended that the growth curve of each transformant
should be measured before performing the experiment described.
In BY4741 wild type strains, it may take about 26 h to reach the
late log phase when using freshly transformed cells with the plas-
mids described here. It is important to treat the cell culture media
gently to prevent leaking of the intracellular proteins.
4. It is important to confi rm that the cells are not precipitated
during cultivation.
5. Centrifugation of culture media will take time (more than 4 h).
Place all other samples on ice during the procedure to avoid
protein degradation.
6. The lyophilized extracellular protein samples can be stored at
−80 °C for at least 1 month.
7. The volume of the 1× buffer used for SDS-PAGE should be
altered if the concentration of proteins is too high. Sometimes,
the amount of positive control protein may increase to such an
extent that secretion of the target proteins may not be detected.
8. The intracellular protein samples can be stored at −80 °C for at
least 1 month. Avoid repeated freezing and thawing (no more
than twice). It is recommended that after extraction, the pro-
tein solution should be immediately used for SDS-PAGE.
