2 Materials
Prepare all solutions using ultrapure water (such as mQ water from
Millipore or equivalent) and analytical grade reagents. When preparing stock solutions and reagents, care should be taken to avoid
keratin contamination (essentially: always use gloves, single-use
plastic ware, and do not touch yourself).
2.1 Preparation
of Plasmids 1
1. DNA restriction enzymes.
2. T4 DNA ligase or DNA ligation kit.
3. Polymerase chain reaction (PCR) reagents.
4. Miniprep/Midiprep kit for plasmid DNA purification.
5. Transformation competent E. coli cells.
2.2 Transfection
of Mammalian Cells
1. Cell line of interest (see Note 1).
2. Cell culture medium (see Note 2).
3. Transfection reagent (see Note 3).
2.3
Proximity-Dependent
Biotinylation
1. Biotin stock solution: 50 mM in neutralized ammonium
hydroxide (see Note 4). Store at À20
C.
2. Doxycycline stock solution: 10 mg mL
À1 in 70% ethanol (see
Note 5). Store at À20
C.
2.4 Streptavidin
Pulldown
1. Streptavidin-coupled magnetic beads (see Note 6). Store at
4
C.
2. Magnet for 1.5-mL tubes.
3. Lysis buffer II: 50 mM Tris–HCl, 500 mM NaCl, 5 mM
EDTA, 0.4% SDS, 1 mM DTT, protease inhibitor cocktail,
pH adjusted to 7.4. Prepare freshly (see Note 7).
4. Equilibration buffer: 50 mM Tris–HCl, 150 mM NaCl, 0.05%
Triton X-100, 1 mM DTT, adjusted to pH 7.4.
5. Wash buffer 1: 2% SDS in water. Prepare freshly (see Note 7).
6. Wash buffer 2: 50 mM Hepes, 1 mM EDTA, 500 mM NaCl,
1% Triton X-100, 0.1% sodium-deoxycholate, pH adjusted to
7.4. Prepare freshly (see Note 7).
7. Wash buffer 3: 10 mM Tris–HCl, 250 mM LiCl, 1 mM EDTA,
0.5% NP-40, 0.5% sodium-deoxycholate, pH adjusted to
8. Prepare freshly (see Note 7).
8. Wash buffer 4: 50 mM Tris–HCl, 50 mM NaCl, 0.1% NP-40,
pH adjusted to 7.4. Prepare freshly (see Note 7).
9. Elution buffer: 10 mM Tris–HCl, 2% SDS, 5%
β-mercaptoethanol, 2 mM biotin, pH adjusted to 7.4. Store
at À20
C.
306
Cinthia Amaya Ramirez et al.
Prepare all solutions using ultrapure water (such as mQ water from
Millipore or equivalent) and analytical grade reagents. When preparing stock solutions and reagents, care should be taken to avoid
keratin contamination (essentially: always use gloves, single-use
plastic ware, and do not touch yourself).
2.1 Preparation
of Plasmids 1
1. DNA restriction enzymes.
2. T4 DNA ligase or DNA ligation kit.
3. Polymerase chain reaction (PCR) reagents.
4. Miniprep/Midiprep kit for plasmid DNA purification.
5. Transformation competent E. coli cells.
2.2 Transfection
of Mammalian Cells
1. Cell line of interest (see Note 1).
2. Cell culture medium (see Note 2).
3. Transfection reagent (see Note 3).
2.3
Proximity-Dependent
Biotinylation
1. Biotin stock solution: 50 mM in neutralized ammonium
hydroxide (see Note 4). Store at À20
C.
2. Doxycycline stock solution: 10 mg mL
À1 in 70% ethanol (see
Note 5). Store at À20
C.
2.4 Streptavidin
Pulldown
1. Streptavidin-coupled magnetic beads (see Note 6). Store at
4
C.
2. Magnet for 1.5-mL tubes.
3. Lysis buffer II: 50 mM Tris–HCl, 500 mM NaCl, 5 mM
EDTA, 0.4% SDS, 1 mM DTT, protease inhibitor cocktail,
pH adjusted to 7.4. Prepare freshly (see Note 7).
4. Equilibration buffer: 50 mM Tris–HCl, 150 mM NaCl, 0.05%
Triton X-100, 1 mM DTT, adjusted to pH 7.4.
5. Wash buffer 1: 2% SDS in water. Prepare freshly (see Note 7).
6. Wash buffer 2: 50 mM Hepes, 1 mM EDTA, 500 mM NaCl,
1% Triton X-100, 0.1% sodium-deoxycholate, pH adjusted to
7.4. Prepare freshly (see Note 7).
7. Wash buffer 3: 10 mM Tris–HCl, 250 mM LiCl, 1 mM EDTA,
0.5% NP-40, 0.5% sodium-deoxycholate, pH adjusted to
8. Prepare freshly (see Note 7).
8. Wash buffer 4: 50 mM Tris–HCl, 50 mM NaCl, 0.1% NP-40,
pH adjusted to 7.4. Prepare freshly (see Note 7).
9. Elution buffer: 10 mM Tris–HCl, 2% SDS, 5%
β-mercaptoethanol, 2 mM biotin, pH adjusted to 7.4. Store
at À20
C.
306
Cinthia Amaya Ramirez et al.