10. Streptavidin Sepharose High Performance affinity resin
(GE Healthcare Life Sciences).
11. 20 mM biotin.
2.4 Sample Digestion
1. Water 18 ohm grade.
2. Formic acid.
3. CH 3 CN, LCMS grade.
4. Hydration buffer: 100 mM of ammonium bicarbonate (AmBi)
5. Washing buffer 1: 100 mM AmBi–CH 3 CN (50:50) (v/v).
6. Washing buffer 2: 25 mM AmBi–CH 3 CN (50:50) (v/v).
7. Reduction buffer: 10 mM DTT in 0.1 M Ambi.
8. Alkylation buffer: 55 mM iodoacetamide in 0.1 M Ambi.
9. Trypsin sequencing grade.
10. 96-well polypropylene plate with conical bottom.
11. Peptide extraction buffer: CH3CN–formic acid 5% (60:40)
(v/v).
3 Methods
3.1 Immunopurification
3.1.1 Mammalian
Expression Vectors
For the identification of TANC1 protein complexes, the TANC1
cDNA was cloned downstream of an N-terminal FLAG tag within
pIRES-puro3 plasmid (see Note 1) using polymerase chain reaction
amplification and standard molecular biology procedures. To identify protein complexes specifically associated with the PDZBM of
TANC1, we mutated the cDNA of TANC1 in order to code for the
AAA sequence instead of the SNV sequence at the carboxyterminus of TANC1.
3.1.2 Verification
of Protein Expression
by Western Blot
Once the sequence integrity is validated, we generally test for
protein expression by western blot analysis following transient
transfections of mammalian cells. Although any mammalian cells
can be used, we routinely use HEK293T cells for this purpose since
they can be efficiently transfected (see Note 2).
1. Grow HEK293T cells in two 10-cm plates up to 40–50%
confluency.
2. For the transfection, mix 5 μg of empty or pIRES-puro vector
expressing the protein of interest with 15 μL of polyethyleneimine (PEI) solution, that is, in a ratio of 1 μg of DNA for 3 μL
of PEI (see Note 3).
3. Probe cell extracts by western blotting for the expression of the
fusion protein using anti-FLAG antibodies.
Identification of Associated PDZ Proteins
21
(GE Healthcare Life Sciences).
11. 20 mM biotin.
2.4 Sample Digestion
1. Water 18 ohm grade.
2. Formic acid.
3. CH 3 CN, LCMS grade.
4. Hydration buffer: 100 mM of ammonium bicarbonate (AmBi)
5. Washing buffer 1: 100 mM AmBi–CH 3 CN (50:50) (v/v).
6. Washing buffer 2: 25 mM AmBi–CH 3 CN (50:50) (v/v).
7. Reduction buffer: 10 mM DTT in 0.1 M Ambi.
8. Alkylation buffer: 55 mM iodoacetamide in 0.1 M Ambi.
9. Trypsin sequencing grade.
10. 96-well polypropylene plate with conical bottom.
11. Peptide extraction buffer: CH3CN–formic acid 5% (60:40)
(v/v).
3 Methods
3.1 Immunopurification
3.1.1 Mammalian
Expression Vectors
For the identification of TANC1 protein complexes, the TANC1
cDNA was cloned downstream of an N-terminal FLAG tag within
pIRES-puro3 plasmid (see Note 1) using polymerase chain reaction
amplification and standard molecular biology procedures. To identify protein complexes specifically associated with the PDZBM of
TANC1, we mutated the cDNA of TANC1 in order to code for the
AAA sequence instead of the SNV sequence at the carboxyterminus of TANC1.
3.1.2 Verification
of Protein Expression
by Western Blot
Once the sequence integrity is validated, we generally test for
protein expression by western blot analysis following transient
transfections of mammalian cells. Although any mammalian cells
can be used, we routinely use HEK293T cells for this purpose since
they can be efficiently transfected (see Note 2).
1. Grow HEK293T cells in two 10-cm plates up to 40–50%
confluency.
2. For the transfection, mix 5 μg of empty or pIRES-puro vector
expressing the protein of interest with 15 μL of polyethyleneimine (PEI) solution, that is, in a ratio of 1 μg of DNA for 3 μL
of PEI (see Note 3).
3. Probe cell extracts by western blotting for the expression of the
fusion protein using anti-FLAG antibodies.
Identification of Associated PDZ Proteins
21