1. Wash the cells carefully in the dish using ice-cold PBS. Carefully
remove the media and wash the cells with 10 mL of PBS.
Repeat this step three times.
2. Add 2 mL of ice-cold lysis buffer per plate. Scrape the cells
using cell scrapers to favor membrane crushing and carefully
collect in 15 mL conical tubes. Tubes are then placed on a
rotator for 30 min to 1 h (see Note 9).
3. To guarantee complete lysis of the cells, proceed to one freeze–
thaw cycle using liquid nitrogen (see Note 10).
4. Thaw the lysates and aliquot them into ten 1.5 mL microfuge
tubes. Spin at 15,000 Â g for 15 min at 4
C. Collect the
supernatant which consist of the soluble fraction into a
15 mL conical tubes (see Note 11).
5. Equilibrate 20 μL of packed ANTI-FLAG
® M2 Affinity agarose
beads by three 1 mL washes with lysis buffer (protein and
phosphatase inhibitors are not required).
6. Spin down beads at 800 Â g for 1 min. To carefully remove the
supernatant and not the beads, we use a clean 27-gauge needle
attached to the vacuum system.
7. Transfer the beads to the 15 mL conical tube containing the
soluble protein fraction and incubate on a rotator overnight at
4
C. We generally prepare the lysates in the afternoon and
incubate them on beads overnight.
8. Spin down at 800 Â g the beads and discard the supernatant.
9. Wash the beads once with 10 mL of lysis buffer and transfer
them into a 1.5 mL microfuge tube to perform 3 additional
washes using 800 μL of lysis buffer (see Note 12).
3.1.7 Elution
Since the presence of detergents is not compatible with MS, it is
important to eliminate detergents. For this purpose either of the
three options listed below can be used.
1. Option A: Elute proteins bound to ANTI-FLAG
® M2 Affinity
agarose beads by using 20 μL of NuPAGE
TM LDS 2Â sample
Buffer
2. Heat the beads at 70
C for 10 min.
3. Load 10% of the eluted fraction on an electrophoresis
NuPAGE
® pre-cast gel system (4–12%).
4. After migration, gels are silver stained using protocols compatible with mass spectrometer [7] or using commercially available
kit such as Pierce™ Silver Stain Kit (Fig. 3a).
5. Option B: Elute proteins using 3xFLAG
® peptide using five
column-volumes (~100 μL) solution at 100 μg/mL in 50 mM
ammonium bicarbonate, pH8.0.
Identification of Associated PDZ Proteins
23
remove the media and wash the cells with 10 mL of PBS.
Repeat this step three times.
2. Add 2 mL of ice-cold lysis buffer per plate. Scrape the cells
using cell scrapers to favor membrane crushing and carefully
collect in 15 mL conical tubes. Tubes are then placed on a
rotator for 30 min to 1 h (see Note 9).
3. To guarantee complete lysis of the cells, proceed to one freeze–
thaw cycle using liquid nitrogen (see Note 10).
4. Thaw the lysates and aliquot them into ten 1.5 mL microfuge
tubes. Spin at 15,000 Â g for 15 min at 4
C. Collect the
supernatant which consist of the soluble fraction into a
15 mL conical tubes (see Note 11).
5. Equilibrate 20 μL of packed ANTI-FLAG
® M2 Affinity agarose
beads by three 1 mL washes with lysis buffer (protein and
phosphatase inhibitors are not required).
6. Spin down beads at 800 Â g for 1 min. To carefully remove the
supernatant and not the beads, we use a clean 27-gauge needle
attached to the vacuum system.
7. Transfer the beads to the 15 mL conical tube containing the
soluble protein fraction and incubate on a rotator overnight at
4
C. We generally prepare the lysates in the afternoon and
incubate them on beads overnight.
8. Spin down at 800 Â g the beads and discard the supernatant.
9. Wash the beads once with 10 mL of lysis buffer and transfer
them into a 1.5 mL microfuge tube to perform 3 additional
washes using 800 μL of lysis buffer (see Note 12).
3.1.7 Elution
Since the presence of detergents is not compatible with MS, it is
important to eliminate detergents. For this purpose either of the
three options listed below can be used.
1. Option A: Elute proteins bound to ANTI-FLAG
® M2 Affinity
agarose beads by using 20 μL of NuPAGE
TM LDS 2Â sample
Buffer
2. Heat the beads at 70
C for 10 min.
3. Load 10% of the eluted fraction on an electrophoresis
NuPAGE
® pre-cast gel system (4–12%).
4. After migration, gels are silver stained using protocols compatible with mass spectrometer [7] or using commercially available
kit such as Pierce™ Silver Stain Kit (Fig. 3a).
5. Option B: Elute proteins using 3xFLAG
® peptide using five
column-volumes (~100 μL) solution at 100 μg/mL in 50 mM
ammonium bicarbonate, pH8.0.
Identification of Associated PDZ Proteins
23