Peptide pulldown with Fc-tagged ECD proteins

This protocol is written as performed for IPs of Fc-tagged ECD protein with 7 serial dilutions of peptide substrate (7.5x master mix of Dynabeads G+protein bait).

1. Prepare master mix of Fc-tagged ECD protein conjugated to Dynabeads G
   1. Cut the pipette tip for a wider opening to withdraw and expel beads.
   2. Wash 100 μl Dynabeads G (ThermoFisher 10007D) with 300 μl 1xPBS x 3 at RT through bead separation on a magnetic rack.
   3. Removed wash buffer from Dynabeads G.
   4. Incubate 150 μg of Fc-tagged ECD protein with Dynabeads G in 500 μl of 1xPBS at RT for 1-1.5 hr with rotation.
   5. Remove excess Fc-tagged protein and washed with 1 ml of 1x PBS x 2
   6. Wash with 1 ml of the reaction buffer (20 mM HEPES pH 7.2, 100 mM KOAc, 0.2% Tween-20. Tween-20 is important to prevent non-specific adherence of peptide)
   7. Resuspend in Dynabeads G-protein mix in 375 μl of the reaction buffer
2. With cut, wide-opening tip, distribute 50 μl of the bead-protein mix into 7 tubes
3. Prepare serial dilutions of peptide substrate
   1. Make separate 60 ul-solutions of 150 μM and 200 μM peptide (measured by Abs at 280 nm) in reaction buffer
   2. Make 2-fold dilutions of peptide for 30 ul each
4. Remove wash buffer from bead-protein mix and immediately add 25 ul of peptide solution to each of the 7 tubes
5. Incubated on rotating shaker at RT for 1 hr
6. Remove unbound peptide and added 50 μl of reaction buffer. Briefly vortex the tube to resuspend the beads for washing.
7. Remove wash and add 25 μl of 1x non-reducing Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2.5% SDS, 10% glycerol) to each bead sample.
8. Incubate at 65C for 15 min to elute protein.
9. Collect sample buffer and add 5 μl of 1x Laemmli buffer with 100 mM DTT for 30 μl total and boil at 95C.
10. Load 20 μl of each sample onto a 12% Genscript ExpressPlus PAGE gel run at 130V for 45 min and stained with Coomassie blue.

Notes:

• Bands for DR5 and TNFR1 ECD proteins appear wide due to glycosylation
• The peptide used (expected MW 2 kDa) migrates at ~10 kDa because small peptides and proteins < 20 kDa exhibit poor resolution in Laemmli-SDS-PAGE (Schägger, 2006).

Reference:

Schägger H. 2006. Tricine-SDS-PAGE. Nat Protoc 1:16–22. doi:10.1038/nprot.2006.4

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