Biomedical Engineering Reference
In-Depth Information
1. Resuspend endosomal membranes in HB buffer. Volume is
dependent on the number of reactions (10
3.4 Reaction
Conditions
ʼ
L for control reac-
tions, and 15
L per experimental reaction). Example: For
seven experimental reactions add 115
ʼ
ʼ
L of HB.
2. Starting material controls.
(a) Add 5
L of endosomal membranes (starting material) to
a microcentrifuge tube and place on ice (no incubation)
for 3 h (Fig.
1a
). Skip ahead to step 7.
(b) Add 5
ʼ
L of endosomal membranes (starting material) to
a microcentrifuge tube and place on ice for 3 h (no incu-
bation) (Fig.
1b
). Then add 6
ʼ
ʼL
of Trypsin (0.27
ʼ
g/
ʼ
L)
and place on ice for 30 minutes. Move to step 7.
(c) For additional controls
see
Note 4
.
3. For experimental reactions (Fig.
1c
):
(a) For the mammalian cytosol reactions add 15
ʼ
L mem-
branes, 6
ʼ
L ATP regeneration system, 25
ʼ
g of HeLa
L (Fig.
2a
).
(b) For the
Saccharomyces cerevisiae
cytosol reactions, add
15
cytosol, and HB up to 50
ʼ
ʼ
L membranes, 6
ʼ
L ATP regeneration system, 70
ʼ
g
of
Saccharomyces cerevisiae
cytosol, and HB up to 50
ʼ
L
(Fig.
2b, d
).
(c) For the
Drosophila melanogaster
(fl y) cytosol reactions,
add 15
ʼ
L membranes, 6
ʼ
L ATP regeneration system,
25
ʼ
g of
Drosophila melanogaster
cytosol, and HB up to
L (Fig.
2c
).
(d) For the experimental reactions immunodepleted of Hrs,
incubate the membranes with a monoclonal antibody
against Hrs (2
50
ʼ
g per reaction) for 30 min on ice followed
by centrifugation. Additionally, immunodeplete Hrs from
cytosol by incubation with a monoclonal anti-Hrs anti-
body for 1 h at 4 °C. Add 15
ʼ
ʼ
L of membranes, 6
ʼ
L ATP
regeneration system, 25
g of Hrs-immunodepleted HeLa
cytosol, and HB up to 50
ʼ
L (Fig.
3
).
(e) For the experimental reactions immunodepleted of Hrs
plus recombinant Hrs protein, incubate the membranes
with a monoclonal antibody against Hrs (2
ʼ
g per reac-
tion) for 30 min on ice followed by centrifugation.
Additionally, immunodeplete Hrs from cytosol by incuba-
tion with a monoclonal anti-Hrs antibody for 1 h at
4 °C. Add 15
ʼ
ʼ
L of membranes, 6
ʼ
L ATP regeneration
system, 25
g of Hrs-immunodepleted HeLa cytosol,
180 nM of recombinant Hrs protein, and HB in a fi nal
reaction volume of 50
ʼ
L (Fig.
3
).
4. Incubate the experimental reactions for 3 h at 37 °C.
See
Note 5
.
5. After incubation, place the reactions on ice and add 6
ʼ
ʼ
L tryp-
sin (0.27
ʼ
g/
ʼ
L). Incubate for 30 min.
See
Note 6
.
