Materials and Reagents

HeLa cells (ATCC, catalog number: CCL-2 )
SBP-Calmodulin Tag Expression Vector pNTAP (Inter Play N-Terminal Mammalian TAP Vector, Agilent Technologies-Stratagene, catalog number: 240101 )
Lipofectamine (Life Technologies, Invitrogen^TM, catalog number: 11668-027 )
Fugene 6 (F. Hoffmann-La Roche, catalog number: 05061377001 )
FLAG tag Expression Vector pCMV-Tag2 (Agilent Technologies-Stratagene, catalog number: 211172 )
NP40
Steptavidin agarose beads (Sigma-Aldrich, catalog number: S-1638 )
Anti- Calmodulin antibody (Upstate, catalog number: 07-482 )
Anti- Flag antibody (Sigma-Aldrich, catalog number: F1804-200UG )
Protease inhibitor cocktail, EDTA free (F Hoffmann-La Roche, catalog number: 04693159001 )
DNase I (Sigma-Aldrich, catalog number: DN25 )
Benzonase nuclease(Sigma-Aldrich, catalog number: E1014 )
Laemmli loading buffer (see Recipes)
Sucrose buffer (see Recipes)
Nuclei Lysis buffer for hard to extract nuclear factors (see Recipes)
Nuclei Lysis buffer for soluble nuclear factors (see Recipes)

Equipment

Wheel in a cold room
Refrigerating centrifuge 1.5 ml tubes

Procedure

Cell transfection
1. Co-transfected 10 to 50 x 10⁶ of HeLa cells (or other “easy to transfect cells”) with your gene of interest “1”-FLAG cloned into pCMV-Tag2 expression vector, and your gene of interest “2”-SBP-Calmodulin cloned into pNTAP, or the empty expression vector pNTAP as a control. Transfection is performed with standard transfection protocols (such as Lipofectamine, Invitrogen or Fugene 6, Roche or Calcium Chloride precipitation).

Nuclear extract preparation
24 to 48 H after transfection, the cells are lysed and nuclear extracts were prepared using the nuclear extract lysis protocol:
1. Use trypsin to recover the cells and wash cells with cold 1x PBS then centrifuge for 6 min at 300 x g at 4 °C. From now on, all the steps should be performed on ice.
2. Resuspend the cell pellet in the chilled Sucrose buffer (5 μl/1 x 10⁶ cells).
3. Add vol/vol Sucrose buffer containing 0.5% NP40 (final concentration 0.25%).
4. Mix by pipetting on ice. Save a small aliquot (1 to 5% -aliquot 1).
5. Centrifuge 10 min at 1,100 x g at 4 °C. The pellet contains the nuclei and looks nacreous to white. The supernatant contains the cytoplasmic protein extract and can be saved if a cytoplasmic protein is of interest for Co-IP. Save a small aliquot (1 to 5% -aliquot 2).
6. Wash the pellet with Sucrose buffer (without NP40) and centrifuge 10 min at 2,000 rpm at 4 °C.
7. Depending on the nuclear proteins to be purified, you can lyse nuclei with:
  1. Either the Nuclei Lysis buffer for soluble proteins (5 μl/1 x 10⁶ cells);
  2. Or the Nuclei Lysis buffer for hard-to-extract proteins (5 μl/1 x 10⁶ cells) and add DNase 5 U/μl and Benzonase 5 U/μl. Resuspension is hard since it’s very viscous with DNA.
8. Incubate on a wheel at 4 °C for 45 min to 1 h. Save a small aliquot (1 to 5% -aliquot 3).
9. Centrifuge 3 min 10,000 x g at 4 °C. The supernatant is the protein nuclei extract that will serve for the IP. Save a small aliquot from the supernatant (1 to 5% -aliquot 4; which is also the input of the IP experiment) and the pellet is the insoluble substance such as membrane debris. Resuspend the pellet in 5 μl/1 x 10⁶ cells of 150 mM NaCl, 10 mM Tris. Save a small aliquot (1 to 5% -aliquot 5).
10. Verify the efficiency of the lysis by analyzing by SDS-PAGE and western-blot the presence of your proteins of interest in the saved aliquots.
  1. Aliquot 1: total cells
  2. Aliquot 2: cytoplasm protein extract
  3. Aliquot 3: total nuclei extract
  4. Aliquot 4: nuclear protein extract
  5. Aliquot 5: insoluble nuclei extract
Co-Streptavidin Purification
1. Incubate the protein nuclei extract 2 h at 4 °C with 25 μl of streptavidin agarose (bead volume). Optimization is required for efficient SP of your SBP-protein of interest and the quantity of streptavidin beads may vary according to the protein stability, etc.
2. Wash 4 to 6 times the beads in 100 mM NaCl, 10 mM Tris-HCl (pH 7.8), by mixing by pipetting and centrifuging 10,000 x g for 30 sec.
3. Elute the bound proteins in Laemmli loading buffer and separate by SDS-PAGE and analyze by western blot using antibodies against Calmodulin and Flag.

Recipes

Laemmli buffer
20% Glycerol
4% SDS
250 mM Tris (pH 6.8) (stacking buffer for upper gel of SDS PAGE)
1.4 M 2-mercapthoethanol
A pinch of bromophenol blue
Sucrose buffer
0.32 M Sucrose
3 mM CaCl₂
2 mM MgOAc
0.1 mM EDTA
10 mM DTT
0.5 mM PMSF
Nuclei Lysis buffer for hard to extract nuclear factors
50 mM Hepes (pH 7.8)
3 mM MgCl₂
300 mM NaCl
1 mM DTT
0.1 mM PMSF
Protease inhibitor Complete mini EDTA free tablets 1x
Nuclei Lysis buffer for soluble nuclear factors
50 mM Hepes (pH 7.8)
50 mM KCl
300 mM NaCl
0.1 mM EDTA
10% Glycerol
1 mM DTT
0.1 mM PMSF
Protease inhibitor Complete mini EDTA free tablets 1x

Notes

The number of cells to be transfected need to be optimized, depending on the size and the stability of the proteins to be expressed.

Acknowledgments

Work in the PF laboratory is supported by institutional grants from 'Institut National de la Santé et de la Recherche Médicale' (Inserm) and 'Centre National de la Recherche Scientifique' (CNRS), and by dedicated grants from the Commission of the European Communities, the 'Agence Nationale de la Recherche' (ANR), the 'Institut National du Cancer' (INCa), the 'ITMO Cancer Alliance Nationale pour les Sciences de la Vie et de la Santé' (AVIESAN) and the 'Fondation Princesse Grace de la Principauté de Monaco'. S.D. was supported by fellowships from the ‘Ministère de l’Enseignement Supérieur et de la Recherche’, the ‘Fondation pour la Recherche Médicale’ (FRM), and the ‘Société Française d’Hématologie’ (SFH).

References

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