Berglund:GST pulldown example

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GST PULL DOWN

USING PURIFIED PROTEIN
IPP100 (20mM Tris pH 8.0, 100mM NaCL, .05% NP-40 =Ipegal CA-630)
IPP150 (20mM Tris pH 8.0, 150mM NaCl, .05% NP-40 =Ipegal CA-630)
IPP200 (20mM Tris pH 8.0, 200mM NaCl, .05% NP-40 =Ipegal CA-630)


1. Incubate GST-fusion protein (5-10uM) with input protein (2.5-5 times more concentrated) together in 200uL total of IPP150. Incubate on ice for 90 minutes
2. Wash 100uL of GST slurry three times with 200uL of IPP150
3. Combine protein and beads and incubate for one hour on rotator at 4C
4. Wash three times with IPP150
5. Run on SDS-PAGE (10uL of beads and 10uL of 2X dye)

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