Preparing beads

for the protein L family:

1. mix 100 ml beads with 30 ml biotinylated protein G (siliconized eppendorf tubes can be used), bring volume to 500 ml with T-TBS+BSA
rock 30 min. @ RT
2. wash the beads 4x with ~800 ml T-TBS+BSA
3. resuspend beads in ~500 ml T-TBS+BSA, add 30 ml whole human IgG (IgG may have to be ultracentrifuged 100,000 x g for 20 minutes to remove aggregation which will help reduce background levels.)
rock 60 min. @ RT
4. wash the beads 4x with ~800 ml T-TBS
5. resuspend beads in 500 ml T-TBS

for the SH3 domain family:

1. mix 100 ml beads with 300 ml soluble Xa-I/Pro cell lysate + 2 ml Tween (siliconized eppendorfs can be used)
rock 60 min. @ RT
2. wash the beads 6x with ~800 ml T-TBS
3. resuspend beads in 500 ml T-TBS

Store beads in the fridge.

Panning

1. remove 15 ml beads for each phage sample to be panned (siliconized eppendorf tubes are recommended to reduce background levels and to reduce the amount of wash steps)
2. wash 2x in ~800 ml T-TBS

Note: bead clumping and stickiness may indicate insufficient Tween. Try higher percentages of Tween in TBS.

3. add desired number of phage to beads, taking the final volume to a minimum of 200 ml in T-TBS; if volume of phage added exceeds 100 ml, add and equal volume T-TBS
4. rock 60 min. @ RT
5. wash (see next page) 7x with ~800 ml T-TBS ; 4x if using siliconized eppendorfs
6. add 200 ml elution buffer to the beads
7. rock 10 min. @ RT
8. give beads a quick vortex then spin down and transfer SN to a new tube
9. neutralize low pH by adding 7 ml 2M Tris base (un-pH'd)
10. infect cells per usual; titration may be necessary

*A note on washing beads:
The technique for washing the beads in T-TBS is subjective. A proven technique is the following:

- quick spin the beads to the bottom of the tube
- tilt the eppendorf tube ~70 degrees off vertical, bringing the meniscus close to, but not touching, the tube lid
- slowly slide the bead pellet up the side of the tube with the magnet until the magnet hits the flange of the tube lid; leave the magnet in place to hold the bead pellet
- tilt the tube back to vertical, allowing the meniscus to pass gently over the bead pellet
- quickly open the tube and remove the rinse solution by aspirator; add T-TBS
- mix the beads/T-TBS by inverting the tube 6 or 8 times; vortex mixing is not necessary

NOTE: do NOT allow the beads to dry. Once the beads contact air, you must proceed quickly. Also, aspirate at low suction, as aspiration facilitates bead drying.

Solutions

T-TBS+BSA is 0.5% Tween, 0.1% BSA in 1x TBS

T-TBS is 0.5% Tween in 1x TBS

Elution buffer is 1:1 mixture of BSA (2mg/ml) and 0.2M Glycine @ pH 2.4