Biomedical Engineering Reference
In-Depth Information
Bradford assay. For cytosol preparation from yeast deletion
strains
see
Note 3
.
3.
Drosophila melanogaster
cytosol preparation:
Drosophila mela-
nogaster
strains should be grown in bottles on standard labora-
tory fl y food. Collect fl ies at desired ages, weigh them as
collected, and freeze at −80 °C. When at least 1 g of fl ies has
been collected, consolidate the fl ies, freeze in liquid nitrogen,
and vortex repeatedly (fl y heads, bodies, and appendages will
be broken apart), making sure that fl ies remain frozen and do
not warm up (or they will stick together). Pour frozen fl ies
through two stacked sieves (No. 25 and No. 48); make sure
that sieves are pre-frozen or cooled with liquid nitrogen so that
fl ies do not stick to the sieves. The No. 25 sieve will isolate
bodies, the No. 48 sieve will isolate heads, and legs will fall
through to the bottom. Without letting them defrost, pour
heads into a glass-glass homogenizer. Homogenize heads in
~1 mL of fl y buffer on ice. Spin homogenate at 1,660 ×
g
in
microfuge to pellet chiton; save supernatant. Homogenize chi-
ton in buffer and centrifuge again at 1,660 ×
g
and recover
additional supernatant. Supernatants should be combined, ali-
quoted, fl ash frozen in liquid nitrogen, and stored at −80 °C
until ready to use. Calculate protein concentration using a
Bradford assay.
3.3 Isolating
Endosomal
Membranes
1. Grow HeLa cells on 10 cm plates to 75-80 % confl uence.
2. Before harvesting, serum starve cells for 2 h by washing cells
with 6 mL of DMEM twice, and then adding 4 mL of starva-
tion media to each plate and placing at 37 °C in 5 % CO
2
for
2 h.
3. After 2 h, stimulate cells for 10 min at 37 °C with warm
100 ng/mL of EGF.
4. Place cells on ice, aspirate supernatant and wash with 5 mL of
ice-cold PBS. Repeat two times.
5. Aspirate supernatant, and add 1 mL of ice-cold PBS to one
plate. Scrape the cells from the plate and transfer to the next
plate. Repeat until all plates have been scraped and all cells
have been collected.
6. Centrifuge the cells at 1,500 ×
g
for 10 min at 4 °C.
7. Resuspend the cell pellet in 100
L of HB.
8. Lyse the cells by drawing them through a 30-gauge needle
into a 1 mL syringe 30 times within approximately 6 min.
9. Centrifuge the lysate at 800 ×
g
for 5 min to remove debris.
10. Collect the supernatant and centrifuge for 15 min at 1,500 ×
g
.
11. Aspirate the supernatant and recover endosomal membranes
to be used for reconstitution reactions.
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