Phosphoinositides Coated Beads Binding Assay

Materials and Reagents

1. PIP-Beads (Echelon) with the corresponding phosphoinositide of interest. Echelon provides a wide range of PI and other phospholipid coated beads on demand, including a sample pack that has a wide range of PIP-coated beads (P-B00S).
   
2. 2 µg of purified GST-tagged protein per tube of beads and GST alone for control
   The protein can be produced and purified by standard methods of GST-tagged protein purification by glutathione-sepharose beads. For the present assay, it is recommended to elute the protein from glutathione-sepharose beads and quantify it before use. Moreover, it is important to use high concentrations of protein, in order to use the smallest possible volume.
   
3. HEPES
   
4. Lgepal CA630
   
5. SDS
   
6. Glycerol
   
7. Tris-HCl
   
8. β-mercaptoethanol
   
9. Bromophenol blue
   
10. Wash/binding buffer (see Recipes)
    
11. 2x Reducing Laemmli Sample Buffer (see Recipes)
    

Equipment

1. Refrigerated centrifuge
   
2. Oscillatory or gyratory shaker
   
3. Aspiration pump
   
4. Needles 0.7 x 30 mm
   
5. 1.5 ml Tubes
   

Procedure

1. Before starting
   a.Prepare or thaw the purified GST-protein.
   b.Pre-cool the centrifuge.
   c.Prepare the wash/binding buffer.
   d.Make aspiration tips by cutting the tip of a needle with pliers or a similar tool (Figure 1). This will minimize the needle diameter.
   
   
   Figure 1. Aspiration tips
   
   
2. Use 50 µl of PI-beads per protein. Echelon provide beads suspended in buffer 50/50.
3. Briefly wash three times the beads with 500 μl of precooled wash/binding buffer at 300 x g, 30 sec.
4. Centrifuge the beads at 300 x g, 4 °C.
5. Add 1 ml of wash/binding buffer to each tube with PI-beads.
6. Add 2 µg of the interest GST-tagged protein to the PI-beads.
7. Incubate the GST-tagged protein or protein domains with the beads rotating for 2 h at room temperature. For some protein domains longer incubation time or overnight 4 °C incubation may be required, depending on the affinity of the selected domain for the phosphoinositide.
8. Wash the beads by pelleting them by centrifugation (300-400 x g at 4 °C). Then add 1 ml of wash buffer and resuspend the beads. Invert the tube 5 times and centrifuge at 4 °C, 300-400 x g. Aspirate carefully the supernatant, without touching the beads with the tip of the needle. Repeat this process 5 times.
9. After last wash, dry the beads by aspiration and immediately add 100 μl of 2x Laemli buffer.
10. Boil the sample at 95 °C for 5-10 min.
11. The sample now can be kept at -20 °C upon analysis in SDS-PAGE.
12. Centrifuge the samples 5 min at 27,000 x g before loading in SDS-PAGE gel. This will pellet the beads.
13. The binding can be analyzed by western-blot, detecting the interest protein or domain by anti-GST antibody. GST control alone should not produce any kind of binding.

Recipes

1. Wash/binding buffer
   10 mM HEPES (pH 7.4)
   150 mM NaCl
   0.25 % Igepal CA630
   
2. 2x Reducing* Laemmli Sample Buffer
   4% SDS
   20% Glycerol
   120 mM Tris-HCl (pH 6.8)
   5% β-mercaptoethanol
   0.2% Bromophenol blue
   *It is important that the buffer is Reducing and fresh, because the reducing agent together with the boiling will be necessary for eluting the protein from the phosphoinositides beads.
   

Acknowledgments

This protocol has been adapted from Galvez-Santisteban et al. (2012).

References

1. Di Paolo, G. and De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature 443(7112): 651-657.
   
2. Galvez-Santisteban, M., Rodriguez-Fraticelli, A. E., Bryant, D. M., Vergarajauregui, S., Yasuda, T., Banon-Rodriguez, I., Bernascone, I., Datta, A., Spivak, N., Young, K., Slim, C. L., Brakeman, P. R., Fukuda, M., Mostov, K. E. and Martin-Belmonte, F. (2012). Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells. Nat Cell Biol 14(8): 838-849.
   
3. Martin-Belmonte, F. and Mostov, K. (2007). Phosphoinositides control epithelial development. Cell Cycle 6(16): 1957-1961.
   
4. Szentpetery, Z., Varnai, P. and Balla, T. (2010). Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling. Proc Natl Acad Sci U S A 107(18): 8225-8230.