1. David Bui Randall Mello Richard Lauhead 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

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

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 E. coli solution and resuspended in 30 mL of Resuspension buffer Column purification protocol involves 10mL of lysate poured into a column with 500 µL of agarose nickel bead solution with subsequent 10 mL washes. Elution took place 500 µL at a time and continued until the beads showed no fluorescence. Wash solutions adapted from Qiagen Ni-NTA agarose beads purification booklet

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

8.  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

9. Ypet-UBC9 could be around 70% pure Cypet-SUMO1 is unlikely to be 97% pure Ypet-UBC9Cypet-SUMO1 Prot.1Prot.2Prot.3 Prot.1Prot.2Prot.3

10. Keeping a constant fluorescent protein amount at 1 µg. BL21 cell lysate proteins were added to change percent purity. Results show that purity has little effect on fluorescence at 1µg of fluorescent protein. Other Proteins do not interfere with fluorescent intensity

11. 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/µl). 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. Purity does not have significant effect on FRET ratio

12.  Experimental Design  Lyophilize 1mL of protein solution in 1.5mL tubes for Cypet-SUMO1 and Ypet-UBC9 for the following tests  Lyophilized and stored as powder at RT  Lyophilized and stored as powder at 4 degree  Lyophilized and stored as powder at -20 degree  Lyophilized and stored as powder at -80 degree  Lyophilized then stored as solution at RT  Lyophilized then stored as solution at 4 degree  Lyophilized then stored as solution -20 degree  Lyophilized then stored as solution -80 degree

13.  Obtained purity before lyophilization  Weighed tube before and after lyophilization to obtain protein amount and fluorescent protein amounts  Diluted all solutions to have the same protein concentrations, and same total protein amount per well.  Performed FRET analysis of each solution to determine FRET effeciency

16. 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 optimization1/18/20102/8/2010100% Flexstation 2 fluorescence sensitivity test1/18/20102/8/2010100% Dialysis/Lyophilization2/1/20102/22/2010 100% Protein purity effects2/1/20102/22/201050% Expression optimization3/1/20103/15/2010 Fret sensitivity3/1/20103/15/2010 10% compound screening sensitivity3/22/20104/5/2010 Stability testing3/22/20104/5/2010 10% Oxidation testing4/12/20104/26/2010 Kit assembly4/12/20104/26/2010 Add secretion factors to proteins5/3/20105/31/2010