Template:SBB-Protocols Assay2

Strepavidin-Binding Assay

Goals: 
1) To Screen 16 constructs for their ability to bind strepavidin on the cell surface
2) To devise a method for quantifying the relative amount of strepavidin bound by the constructs

For all 16 constructs & the 3 controls: grow them with/without arabinose to saturation in 1-3uL culture (grown in 24 well blocks)
96 well plate:
- each well add 300ul PBS, 15ul Streptavidin, 25ul of cell culture (the first time we do this, it will be saturated culture)
- incubate at 37C without shaking for 30mins-1hour
- spin down cells for 1 min at full speed to pellet cells - check in UV box for bright white color

Controls:
pBca9145-Bca 9494 (positive control that displays cpx, a streptavidin binding peptide under Pbad)
DH10B (no plasmid, negative control)
pBca9495CA-Bca1144 (negative control: backbone)

Can play with:
- streptavidin concentrations
- mid-log vs. saturation
- quantitative assays (number of washes, volume...)

JCA: This needs much more detail. In particular, write up the details about the volumes of materials, composition of the assays, times and temperatures at each step, etc.

April 22

- today we tried to do the assay with 25ul cells in 300ul PBS with 15ul Streptavidin
- we could actually visually see binding for 4 constructs (one control?)
- we tried to quantify it with the Tecan but we just got background

for Monday
- try 200ul cells
- spin down
- discard the media
- resuspend in 200ul PBS
- use 5ul Streptavidin
- incubate at 37 for 30 mins
- spin down, remove supernatant
- check visually
- resuspend in 50 ul of PBS and load on plate for the Tecan
- analyze measurements