11. Distribute the adjusted lysates (total volume approximately
3.3 mL) in three 1.5-mL tubes and clear by centrifugation at
16,000 Â g for 10 min at 4
C.
12. Pool the supernatants from each condition in 15-mL tubes.
13. Determine the protein concentration of each sample using a
standard Bradford assay. Adjust volumes of each sample, so that
they all have equal concentration and keep 10% aside as Input
sample for later analysis. Typically 3–3.5 mg of protein content
is used for a streptavidin pulldown.
3.4 Streptavidin
Pulldown
for Proteomics Studies
1. Pre-equilibrate the total amount of streptavidin-coupled beads,
typically 200 μL per pulldown (see Note 24) by transferring the
beads to a 1.5-mL tube and wash them once with 1 mL equilibration buffer.
2. Dispatch the equilibrated beads in enough 1.5-mL tubes to
accommodate the starting amount of 3–3.5 mg protein sample
per conditions (usually two to four tubes are needed).
3. Place the tubes on a magnetic rack, remove the supernatant,
and for each condition, resuspend the beads with equal
amounts of cleared lysates.
4. Incubate the bead/lysate mixture overnight at 4
C on a rotating wheel.
5. On the next day, place the tubes on a magnetic rack, wait until
the beads have stuck to the side of the tubes and gently aspirate
the supernatants using a capillary tip connected to a vacuum
pump. From now on, the following steps are performed at RT.
6. In each tube, resuspend the beads with 200 μL of wash buffer
1 and pool beads from the same conditions in a single 1.5mL tube.
7. Incubate for 8 min on a rotating wheel.
8. Place the tubes on a magnetic rack, wait until the beads have
stuck to the side of the tubes and gently aspirate the supernatants using a capillary tip connected to a vacuum pump or
manually with a pipette.
9. Wash the beads once more with 1 mL of wash buffer 1 as in
steps 7 and 8.
10. Following the same procedure as in step 9, wash the beads
twice with 1 mL of wash buffer 2, then wash buffer 3, then
transfer to fresh 1.5-mL tubes and proceed with wash buffer 4.
11. After the final wash, briefly centrifuge the samples on a table
micro-centrifuge, place back on the magnetic rack, and remove
any remaining buffer with a pipette.
12. Elute captured biotinylated proteins (see Note 25) by adding
30 μL of elution buffer to each sample. Incubate at 98
C,
312
Cinthia Amaya Ramirez et al.
3.3 mL) in three 1.5-mL tubes and clear by centrifugation at
16,000 Â g for 10 min at 4
C.
12. Pool the supernatants from each condition in 15-mL tubes.
13. Determine the protein concentration of each sample using a
standard Bradford assay. Adjust volumes of each sample, so that
they all have equal concentration and keep 10% aside as Input
sample for later analysis. Typically 3–3.5 mg of protein content
is used for a streptavidin pulldown.
3.4 Streptavidin
Pulldown
for Proteomics Studies
1. Pre-equilibrate the total amount of streptavidin-coupled beads,
typically 200 μL per pulldown (see Note 24) by transferring the
beads to a 1.5-mL tube and wash them once with 1 mL equilibration buffer.
2. Dispatch the equilibrated beads in enough 1.5-mL tubes to
accommodate the starting amount of 3–3.5 mg protein sample
per conditions (usually two to four tubes are needed).
3. Place the tubes on a magnetic rack, remove the supernatant,
and for each condition, resuspend the beads with equal
amounts of cleared lysates.
4. Incubate the bead/lysate mixture overnight at 4
C on a rotating wheel.
5. On the next day, place the tubes on a magnetic rack, wait until
the beads have stuck to the side of the tubes and gently aspirate
the supernatants using a capillary tip connected to a vacuum
pump. From now on, the following steps are performed at RT.
6. In each tube, resuspend the beads with 200 μL of wash buffer
1 and pool beads from the same conditions in a single 1.5mL tube.
7. Incubate for 8 min on a rotating wheel.
8. Place the tubes on a magnetic rack, wait until the beads have
stuck to the side of the tubes and gently aspirate the supernatants using a capillary tip connected to a vacuum pump or
manually with a pipette.
9. Wash the beads once more with 1 mL of wash buffer 1 as in
steps 7 and 8.
10. Following the same procedure as in step 9, wash the beads
twice with 1 mL of wash buffer 2, then wash buffer 3, then
transfer to fresh 1.5-mL tubes and proceed with wash buffer 4.
11. After the final wash, briefly centrifuge the samples on a table
micro-centrifuge, place back on the magnetic rack, and remove
any remaining buffer with a pipette.
12. Elute captured biotinylated proteins (see Note 25) by adding
30 μL of elution buffer to each sample. Incubate at 98
C,
312
Cinthia Amaya Ramirez et al.