Concanavalin A enrichment

ConA enrichment

This protocol is based on ConA enrichment on a confluent 10cm dish, seeded with confluent mouse embryonic fibroblasts.

Step One: Cell lysis

1)     Wash cells twice in ice-cold PBS. 

2)     Lyse cells on ice for 10 minutes in ice-cold TX-100 lysis buffer (1% Triton X-100, 150mM NaCl, 50 mM Tris-HCl, pH 7.4) containing complete protease inhibitor cocktail (Roche) and 10 mM 1,10-phenanthroline, (this prevents ADAM17 autocatalysis). 

3)     Gently move/swirl the dishes during the 10 minute lysis period, to optimize lysis of the whole monolayer.  

4)     Harvest the lysate into an eppendorf using a cell scraper and clarify by spinning at 13,000 x g at 4°C in a bench-top centrifuge.  

5)     Save a fraction of the clarified lysate (50 µl) as an input to run on the gel alongside the enriched fraction.

6)     (Optional) If comparing conditions that alter cell viability, then measure the protein concentration, using BCA kit. Use 12.5 µl sample + 12.5 µm water to reduce the phenanthroline concentration, as it precipitates in the kit components. As a control use 12.5 ul lysis buffer +12.5 ul water, but also do a second control, containing 25 ul water.

Step Two: Prepare ConA agarose beads

7)     Use 50 ul beads per sample. Wash twice with 1 ml TX-100 lysis buffer, spin at 1000-2000 x g for 1-2 min between washes. Wash the beads in bulk e.g. for three plates, then wash 150 ul beads, and then split the washed beads 1/3 into three eppendorfs.

Step Three: ConA enrichment of lysates

8)     Add the clarified lysates from Step One to the washed ConA beads in Step Two. 

9)     Incubate for 2-3 h at 4°C on a rotor. This gives cleaner results than an overnight incubation, but O/N incubation also works. 

Step Four: Wash and elute ConA beads

10)  Wash 3 x with 1 ml lysis buffer, as previously described. 

11)  For the last wash, spin at 13,000 x g for 1-2 min, remove as much as you can from the supernatant. 

12)  Elute the glycoproteins with 1x reducing loading buffer, supplemented with 15% sucrose (per ml of 1x loading buffer: 250 µl 4x LDS; 50 µl 1M DTT; 300 µl 50% sucrose; 400 µl water) for 15 min at 65'C in a thermomixer (the sucrose knocks the ConA off the lectin). 

13)  Spin at 13,000 x g and transfer the supernatant into a new tube using a flat loading tips, to load on a gel.