COLONY LIFT FOR PROTEIN L FAMILY
A secondary screen for protein L
1. For each plate to be lifted, make one induction plate. Add 1µl IPTG stock to 100µl H2O, spread on plate with appropriate antibiotics.
2. Lay (using gloves) 0.05µm pore nitrocellulose filter over colonies and
push out (w/ forceps) any bubbles. Lift filter (w/ forceps) and transfer to induction plate, colony-side up. Incubate 1 hr. @ 37 C.
3. Wash colonies completely from filter by rubbing off all colonies using gloves under running dH2O.
4. Block filters in 5 mls T-TBS 10 min, RT/gentle shaking.
5. Add 1:4000 dilution human IgG into same blocking soln.; 1 hr, RT/gentle shaking.
6. Wash filter 3x w/ T-TBS.
7. Add 1:3500 dilution AP-conjugated goat anti-human in fresh blocking soln.; 1 hr, RT/shaking.
8. Wash filter 3x w/ T-TBS.
9. Equilibrate filter (~1 min) in 5 ml high pH buffer.
10. Develop filter in same buffer by adding 100µl BCIP + 100µl NBT, shaking occasionally. Color will develop anywhere from 1 to 30 min. If filter begins to turn blue-gray, go to next step.
11. Wash filter with dH2O and leave filter in water for at least 10 minutes before handling to stop rxn. Lay on a paper towel to dry.
Buffer/solution recipes:
T-TBS
0.5% Tween in 1x TBS
High pH buffer
- 100 mM Na2CO3
- 100 mM NaHCO3
- 1 mM MgCl2
or
- 100 mM Tris
- 100 mM NaCl
- 5 mM MgCl2
BCIP is 15 mg/ml in DMF
NBT is 30 mg/ml in 75% DMF
made Feb. 22, 1999; Baker Laboratory
Home |
Overview | Highlights |
Publications | Members | Information |
Links
http://depts.washington.edu/bakerpg