Template:SBB- Streptavidin Display Assay Microscopy

Streptavidin Display Microscopy Assay
NOTE: The microscopy protocol has been superseded by the SafeImager assay. The following is in beta and likely to stay that way.

Positive control is pBca9145-Bca9494 {AraC-Pbad}{rbs.cpx}

Negative control is pBca9495CA-Bca1363 {Pbad.rbs.prepro.StrepTag}

Wells containing (a) 35 ul of cell media and 5 uL PBS and (b) 35 ul of cell media and 5 uL streptavidin in PBS should be included to measure background fluorescence.

1. Grow cells to saturation and induce with arabinose. Typically, inoculate from an existing culture 1:1000 or from a colony and incubate overnight. In the morning, use last night's saturated culture to inoculate 1:100 cell dilution. Add 1:1000 of 100mg/ml arabinose (100μg/mL final concentration)to the new culture and assay in the afternoon (~ 5 hours). Before assaying, measure OD600. [0.2% arabinose also commonly cited for induction- 2 mg/ml or 20X higher] 2. Add 35 uL of cells to wells in a 384-well PCR plate (max volume: 50 μL/well).

3. Centrifuge streptavadin before use to pellet out any aggregates; use only the supernatant. For each sample, add 5 uL of streptavidin (0.04 mg/mL) in PBS to each well (for a final concentration of 0.005 mg/mL). Incubate at 37C without shaking for 30 minutes.

4. Pellet cells by spinning down plate at 5000 RPM for 5 minutes.

5. Perform steps 7-9 below before removing the supernatant (to see if the removal of supernatant is necessary).

6. Remove supernatant, resuspend cells in an equal volume of PBS, and repellet cells.

7. Visualize fluorescence on the Safe Imager and the UV imager, recording each image.

8. View cells under fluorescent microscope, recording the image of each individual well plate.

9. Seal well plate with a film, invert, and visualize cells on the fluorescent microscope whose light source is not in trans. Record each image.

10. Repeat 6-9 (wash steps) until the number of washes is adequate to visualize the results (or the cell loss becomes noticeable).

10. Images will be analyzed with ImageJ.