DotBlot Assay

Dot Blot Assay for Needle scFv binding to espA

constructs: Needle scFv (14total), Gliadin neg control (2), No pass neg control (14), 1363 neg control (1)

do triplets

1. grow cells to saturation overnight (for 12 hours)

2. induce with arabinose. total volume of 600ul. do 1:10 dilution. induce for 5-12hours.

3. add 100ul of cells to Vbottom polystyrene plate. take OD

4. spin down cells. flick out liquid.

5. add 5ul of espA protein to 95ul of TRIS buffer. resuspend cells in solution.

6. incubate for 1.5 hours.

7. centrifuge cells and wash 3X with TRIS buffer on plate washer

8. take OD on last wash.

9. after 3rd wash, pellet cells, and resuspend in lysis buffer (50ul)

10. heat for 15-20 minutes

. run a BCA assay on the lysate and make total protein equal. use lysis buffer to increase concentration. Before pipetting up lysate, centrifuge the solution to pellet genomic DNA stuff. Also, make a spread sheet of all the amounts of SDS needed to be added to the respective wells to bring to same total concentration. (**note, how do you know exactly how much volume there is in the wells? more than 50ul) BCA calculation...will need to run a very good standard. how to precisely predict the amount of protein in each well??

11. pipet 1ul (using multichannel) onto nitrocellulose paper. allow to dry.

12. add anti-his HRP. and incubate for 1hours (?)

13. visualize with UV (?)