1. David Bui Richard Lauhead Randall Mello Michelle Tran

2.  Goal: Development of FRET based kit to screen compounds that could alter binding between SUMO1 and UBC9. Why is it important to have this kit? Disregulation of the SUMO pathway has been linked to diseases including ovarian carcinoma, melanoma, and lung adenocarcinoma. (Mo and Moschos 2005) http://www.biochem.mpg.de/jentsch/Mueller.html

3.  Bradford Assay:  Total protein concentrations of solution can be calculated using the equation obtained from graph.

4.  Using highly pure proteins, serial dilutions were done to make solutions at different protein concentrations  Values range from 1 ng to 100 ug

5.  For accurate fluorescence measurements of single proteins and conjugations, an assay must have values away from background noise  500 ng is the lowest amount for usable assay conditions. Ypet-Ubc9 (ng) RFU without blank 10000058734866.9855943707.3961961881.18 5000036425642.9836053579.3939276933.18 2500020232692.9820081895.3923343669.18 100007194076.487613824.8879610900.176 50004313948.983672442.6374147272.676 25001905796.6051675790.8871826577.176 1000432355.386416138.231433805.832 500118134.269199872.325247566.004 1008604.36515551.56913429.223 502701.48817229.91211205.072 25371.4263043.3348485.249 10-1422.633-372.6353125.487 5-1634.1921552.0711672.986 2.5-1682.9312221.7782313.75 1-1428.963-136.699965.361 0000 Cypet-Sumo1 (ng) RFU without blank 9150022667111.5923441949.3923460794.73 500009461644.59413937961.3915112699.73 250005334212.5948311063.3879603005.729 100002223233.3442547688.1374388946.729 50001010283.2191294915.3872053190.604 2500493361.313681590.324730195.979 1000135762.875196490.34217703.057 50058105.1172562.40369732.471 1006996.2746824.6167213.549 501396.1757010.9683055.906 255140.7563566.683773.441 10-1246.0452060.112739.592 5-1626.1123063.877168.947 2.5-1058.782058.266429.742 1-465.92913409.479-50.309 0000

6. IngredientsConcentration of Solutions(M) Wash1Protocol 1Protocol 2Protocol 3 NaCL0.30.50.4 Tris HCL pH 7.40.02 Wash2 NaCL0.321.2 Tris HCL pH 7.40.20.02 Triton0.50%2.00%1.25% Wash3 NaCL0.321.2 Tris HCL pH 7.40.20.02 Immidazole0.020.050.035 Elution NaCL0.3 Tris HCL pH 7.40.02 Immidazole0.150.250.2 Resuspension BufferConcentration(M) NaCl0.5 Tris-HCl pH 7.40.2 Immidazole0.005 Cell Lysate obtained from 1 Liter of solution and resuspended in 30 mL of Resuspension buffer was obtained. Column purification protocol involves 10mL of lysate poured into a column with 500 uL of agarose nickel bead solution with subsequent 10 mL washes. Elution took place 500 uL at a time and continued until the beads showed no fluorescence.

7.  Using the Bradford Assay to determine total protein concentration and the fluorescence curves generated from the sensitivity tests, the purity was calculated for each protocol Ypet-UBC9 Purification protocol Bradford concentrations(ng/uL) fluorescent concentration (ng/uL)Purity Protocol177105086.890.66 Protocol2955617.270.65 Protocol355002783.920.51 Cypet-SUMO1 Purification protocol Bradford concentrations(ng/uL) fluorescent concentration (ng/uL)Purity Protocol14844.944708.360.97 Protocol23191.381678.810.53 Protocol34642.163229.120.70

8. Ypet-UBC9 could be around 70% pure Cypet-SUMO1 is unlikely to be 97% pure Ypet-UBC9Cypet-SUMO1

9. Keeping a constant fluorescent protein amount at 1 ug. BL21 cell lysate proteins were added to change percent purity. Results show that purity has little effect on fluorescence at 1ug of fluorescent protein.

10. Tested purity effects on FRET with each protein at a constant amount of 500 ng. Results demonstrate that purity of fluorescent proteins and in FRET has no effect at low concentration(10ng/ul). Emission max of Ypet-Ubc9 over Cypet-SUMO1 to obtain FRET ratio. Results demonstrate little to no change in FRET ratio when purity is varied.

11. SUMO ASSAY KIT 1/18/20101/25/2010 2/1/20102/8/2010 2/15/20102/22/2010 3/1/20103/8/2010 3/15/20103/22/20103/29/2010 4/5/2010 4/12/20104/19/20104/26/2010 5/3/2010 5/10/20105/17/20105/24/20105/31/2010 6/7/2010 TasksStartEnd %comple te Purification optimization1/18/20102/8/201090% Flexstation 2 fluorescence sensitivity test1/18/20102/8/2010100% Dialysis/Lyophilization2/1/20102/22/2010 Protein purity effects2/1/20102/22/201050% Expression optimization3/1/20103/15/2010 Fret sensitivity3/1/20103/15/2010 compound screening sensitivity3/22/20104/5/2010 Stability testing3/22/20104/5/2010 Oxidation testing4/12/20104/26/2010 Kit assembly4/12/20104/26/2010 Add secretion factors to proteins5/3/20105/31/2010