Protocol for Needle Assay
14 constructs for needle scFv and 1 construct from gliadin scFv as negative control
Prepare espA-FITC: do spot test to make sure there is protein. Keep protein on ice.
1. inoculate from stock plates into 1ml of LB AC in 96well block. Grow to saturation. 2. induced into triplicates to total volume of 500ul. dilute culture 1:10 in media (so add 50ul of culture) 3. grow/induce for 5hours (or can vary time) 4. add 100ul of cells into V bottom polystyrene plates. Do triplicates. (keep flame on when doing this in case of contamination, which could grow during incubation) 5. take an initial OD at 600nm in a black flat bottom plate 6. transfer the cells to a V bottom plate and pellet the cells (5500RPM) for 5 minutes 7. resuspend in 5ul of espA-FITC in 95ul PBS solution (the protein concentration looks pretty high in the coomassie spot test, so this should be more than enough protein for binding) 8. incubate for 30minutes at 37C without shaking (also wrapped in foil to prevent photobleaching) 9. pellet cells and flick away supernatant 10.resuspend cells in 100ul 1X PBS, centrifuge, flick. Do 3 washes 11.take picture of pellet after 3rd wash 12.transfer cells to black flat bottom plate and take fluroescence measurement after 3rd wash 13. take final OD
Measurement mode: Fluorescence Excitation wavelength: 483 nm Emission wavelength: 525 nm Excitation bandwidth: 20 nm Emission bandwidth: 20 nm Gain (Manual): 50 Number of reads: 20 FlashMode: High sensitivity Integration time: 40 µs Lag time: 0 µs Plate definition file: COS96ft.pdf Z-Position (Manual): 5100 µm Shake duration (Linear Medium): 5 s Target Temperature: 37 °C Current Temperature: 37 °C