IGEM:Harvard/2006/Adaptamers/Notebook/2006-7-11

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7/11

Shawn and William suggested that for an initial assay, it'd be easy to just stain protein with Coomassie blue. So that's what we attempted.

We ran two 10-20% native polyacrylamide gels at 15 V overnight; the first used some lanes from the Nano group's gel. Visualization to come tomorrow.

The composition of what we added to each line is below; materials were added and incubated for 30 minutes in 2 uL total volume for lanes 1-11, 1-12 prior to dilution to 10 uL including addition of loading dye. Lanes 2-1 to 2-9 were incubated for an hour; longer incubation carried out since DNA basepairing reactions for lanes 2-10 to 2-12 were carried out for 30 minutes using results from 30-minute incubations of lanes 2-1 to 2-9. This incubation time should not be significant.

Materials used: Thrombin: a 2 uM solution of Thrombin. Streptavidin: a 2 uM solution of Streptavidin. Thromb5, Thromb20, Thromb35, Thromb50, Strep5, Strep20, Strep35, Strep50 are described in Plan section above. Bock's buffer is a reaction buffer for DNA-protein binding, used in several papers (see Nano group's page). 10X dye is a few flakes of bromophenol blue + 500 uL Bock's + 500 uL glycerol.

All quantities in uL.

Lane solutions
1-11 1-12 2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 2-9 2-10 2-11 2-12
Thrombin .5 .5 .5 1 1 1
Streptavidin .5 .5 1 1 1
Thromb5 1 1
Thromb20 2
Thromb35 2
Thromb50 2
Strep5 2
Strep20 2
Strep35 2
Strep50 2
4X Bock's Buffer (see Nanostructure page) .5 .5 .5 1 1 1 .5 .5 1 1 1
H2O 1 1
2-2 solution 2
2-3 solution 2
2-4 solution 2
2-7 2
2-8 2
2-9 2
Bock's 1X buffer 7 7 7 7 7 7 7 7 7 7 7 5 5 5
10X dye 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Ran 2 10-20% polyacrylamide gels along with nano group.