To determine the interaction between NCKAP1 and WASF3, GST-fusion protein pulldown assays were performed as described previously (5 (link),31 (link)). A GST-WASF3 (GST-W3) fusion protein was expressed in BL21 bacteria and purified using MagneGST glutathione particles (Promega, Madison, WI). Once the correct size protein was confirmed, using Coomassie Brilliant Blue staining following SDS-PAGE, the immobilized fusion protein was used immediately. Cell lysates from MDA-MB-231 cells that had been transfected with a pCDH-CMV-MCS-EF1-PURO-NCKAP1 construct were incubated in 500 µl of binding buffer (20 mm Tris-HCl, pH 7.5, 140 mm NaCl, 1% Nonidet P-40 and 0.5% BSA) with the GST fusion protein tethered to the glutathione particles for 4 h at 4°C. Precipitates were resolved by SDS-PAGE and analyzed by western blotting.
Protocol detail
NCKAP1-WASF3 Interaction Assay
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Assays
Bacteria
Buffer
Cell
Coomassie brilliant blue
Glutathione
Mda mb 231 cells
Nacl
Nckap1
Nonidet p 40
Promega
Protein
Sds page
Tris
Wasf3
Wasf3 protein
Corresponding Organization : University of Georgia
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Variable analysis
independent variables
- Expression of GST-WASF3 fusion protein in BL21 bacteria
- Binding of NCKAP1 from MDA-MB-231 cell lysates to the GST-WASF3 fusion protein
- Binding buffer composition (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1% Nonidet P-40, 0.5% BSA)
- Incubation time (4 hours) and temperature (4°C)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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