Sanford-Burnham

R a tio (5 2 0 /4 9 0 )x 1 0 0 0 0
Fluorescence Ratio (520/490) x10000
S /B
R a tio (5 2 0 /4 9 0 )x 1 0 0 0 0
S/B
CUL1 neddylation assay development
6 0 0 0
6
6 0 0 0
6
4 0 0 0
4
4 0 0 0
4
1 5 m in
2 0 0 0
3 0 m in
6 0 m in
2
1 5 m in
3 0 m in
6 0 m in
2 0 0 0
1 5 m in
3 0 m in
5 0 m in
2
15 min
30 min
50 min
0
0 5 0 0 1 0 0 0
[flu o -N e d d 8 ] (n M )
0
0 5 0 0 1 0 0 0
[flu o -N e d d 8 ] (n M )
0
0 5 0 1 0 0 1 5 0 2 0 0
[S C F ] (n M )
0
0 50 100 150 200
[SCF] (nM)
4000
9 0 0 0
L u m i4 -T b A b
0 .0 6 2 5
3000
0 nM NAE1
12.5 nM NAE1
50 nM NAE1
200 nM NAE1
6 0 0 0
0 .1 2 5
0 .2 5
2000
0 .5
3 0 0 0
1
1000
2
0
0 10 20 30 40 50
Time (min)
• All assay parameters were optimized
0
0 1 0 2 0 3 0 4 0 5 0
[A T P ] (u M )
• Many reagents tested in “matrix” experiments