sites for interaction with extracted proteins and is thus more efficient in terms of purification. However, peptides have access to the
whole set of extracted proteins regardless of their localization in
cells and thus detect “non-natural” interactions. A big advantage of
the peptide pulldown is that it tells precisely where the partners
bind within the bait (here to its carboxy-terminal sequence). We
recently used this combined approach with great success for numerous proteins harboring PDZ binding motives such as JamC [14],
Fas [15], Angiomotin [16], Vangl2 [17], or Amotl2 [18]. These
approaches are versatile, purify protein complexes in native conditions, and can be used with lysates of cellular or tissue origins.
4 Notes
1. Any vectors can be used. We generally used a backbone encoding for a gene of resistance to select stable cell lines. Our vector
contains an internal ribosome entry site (IRES) encoding for
the expression of the puromycin-resistance gene required for
the establishment of stable cell lines or populations expressing
the fusion proteins.
2. The identification of potential interacting PDZ domain proteins rely on their expression in a given cell line. Depending on
the biological question, it can be suitable to use other appropriate cell lines. We generally work with HEK293T cells since
they are easy to handle and transfect.
3. We recommend using the polyethyleneimine based transfection
procedure, which is inexpensive and efficient for the transfection of HEK293T cells, however, any other transfection
reagent may be used. To avoid false positive interacting proteins and undesired heat shock proteins associating with the
bait protein, use of clones expressing lower levels of the fusion
proteins may help.
4. Overexpression of some proteins or mutant form can lead to
cytotoxicity. To overcome this limitation, we recommend the
use of inducible expression cell system such as Flp-in
TM
T-Rex
TM system (Thermo Fisher Scientific).
5. Half of the sample can be stored in frozen vials or used to
generate cell lysates for western blot analysis of gene expression
to confirm transfection efficiency.
6. Depending on the expression level and purification yield, more
starting materiel may be necessary. For some purification, we
used up to 1 Â 10
9 cells.
7. All buffers should be filtrated with 0.44 μm filters to remove
any trace of dust and keratins.
36
Avais M. Daulat et al.
whole set of extracted proteins regardless of their localization in
cells and thus detect “non-natural” interactions. A big advantage of
the peptide pulldown is that it tells precisely where the partners
bind within the bait (here to its carboxy-terminal sequence). We
recently used this combined approach with great success for numerous proteins harboring PDZ binding motives such as JamC [14],
Fas [15], Angiomotin [16], Vangl2 [17], or Amotl2 [18]. These
approaches are versatile, purify protein complexes in native conditions, and can be used with lysates of cellular or tissue origins.
4 Notes
1. Any vectors can be used. We generally used a backbone encoding for a gene of resistance to select stable cell lines. Our vector
contains an internal ribosome entry site (IRES) encoding for
the expression of the puromycin-resistance gene required for
the establishment of stable cell lines or populations expressing
the fusion proteins.
2. The identification of potential interacting PDZ domain proteins rely on their expression in a given cell line. Depending on
the biological question, it can be suitable to use other appropriate cell lines. We generally work with HEK293T cells since
they are easy to handle and transfect.
3. We recommend using the polyethyleneimine based transfection
procedure, which is inexpensive and efficient for the transfection of HEK293T cells, however, any other transfection
reagent may be used. To avoid false positive interacting proteins and undesired heat shock proteins associating with the
bait protein, use of clones expressing lower levels of the fusion
proteins may help.
4. Overexpression of some proteins or mutant form can lead to
cytotoxicity. To overcome this limitation, we recommend the
use of inducible expression cell system such as Flp-in
TM
T-Rex
TM system (Thermo Fisher Scientific).
5. Half of the sample can be stored in frozen vials or used to
generate cell lysates for western blot analysis of gene expression
to confirm transfection efficiency.
6. Depending on the expression level and purification yield, more
starting materiel may be necessary. For some purification, we
used up to 1 Â 10
9 cells.
7. All buffers should be filtrated with 0.44 μm filters to remove
any trace of dust and keratins.
36
Avais M. Daulat et al.