Biomedical Engineering Reference
In-Depth Information
Mammalian cell transfection was performed as described in the
Lipofectamine™ 2000 (Invitrogen) plasmid DNA transfection
protocol (
see
Note 2
).
3.2.2 Mammalian Cell
Transfection
3.3 Verifi cation
of Expression
It is important to verify that your constructs produce a fusion
protein of expected size (
see
Notes 1
and
3
).
1. Inoculate one yeast colony into 20 mL of liquid Sc-ura-leu
medium and grow overnight with shaking at 30 °C.
2. Dilute cell culture in fresh, warm 20 mL Sc-ura-leu medium to
OD
600
0.2, and regrow to OD
600
0.8-1.0 (
see
Note 4
).
3. Harvest cells by centrifugation at 3,200 ×
g
for 2 min; discard
the supernatant.
4. Resuspend the cells in 1 mL of water, transfer into a 1.5 mL
microfuge tube, and collect by centrifugation.
5. Resuspend the cells in 400
3.3.1 Lysate Preparation
Yeast Lysate Preparation
L 2 % SDS containing the protease
inhibitors (Subheading
2.2.1
), and vortex.
6. Add 200
μ
L of 0.45 mm acid-washed glass beads and heat for
3 min in a 95 °C block heater or equivalent.
7. Vortex twice for 90 s with maximum speed.
8. Centrifuge for 5 min at full speed in a microfuge and collect
the clear lysate in a new tube. Avoid the pellet.
9. Measure the protein concentration by BCA method (BCATM
Protein Assay Kit, Pierce) or equivalent (
see
Note 5
). Typically
a lysate with a protein concentration 8-12
μ
μ
g/
μ
L is obtained.
10. Dilute the lysate to a concentration of 4
L with 6×
Laemmli sample buffer and water to yield 1× sample buffer
concentration.
μ
g/
μ
1. Use 1 day transfected cells grown on a 2 cm dish to about 90 %
confl uence.
2. Wash cell monolayer gently with PBS.
3. Add 1 mL lysis buffer per dish.
4. Break the cells by shearing by pipetting 20× up and down and
rotating the lysate for 15 min at 4 °C.
5. Remove unbroken cells by spinning the lysate for 5 min at
top speed in a microcentrifuge. Collect the supernatant in a
new tube.
6. Measure the protein concentration, and dilute the sample to
1× Laemmli sample buffer as described in Subheading “Yeast
Lysate Preparation” of Subheading
3.3.1.
Mammalian Lysate
Preparation
Apply 20-40
g total protein of every sample to 12 % mini SDS-
PAGE gel. This percentage of gel gives optimal resolution for
20-60 kDa proteins:
μ
3.3.2 SDS-PAGE
