Berglund:Steve Method-ySF1

From OpenWetWare
Jump to: navigation, search

1. Pour a 0.5X TBE, 6% 37.5:1 acrylamide gel and also prepare 0.5X TBE running buffer. Keep the gel and running buffer cold at 4 degrees Celsius.

2. Make a 10X binding buffer (250 mM Tris pH 7.5, 500 mM NaCl, 0.7 mg/mL tRNA, 1% triton X, 10 mM DTT).

3. Make a 10X loading dye solution ( 200 uL 5X TBE, 400 uL 100% glycerol, 50 uL 10% bromophenol blue, 350 uL water).

4. Prepare the RNA as follows: Radiolabel the GAGUAAC stem loop RNA by kinasing with gamma ATP and then gel purify. I usually dilute my purified RNA to about 10 to 20 nM so it is about 10X my desired final concentration, which is about 1 to 2 nM. I try and have my RNA concentration a 100 fold below the lowest protein concentration. Once I have my RNA ready, I heat it at 70 degrees Celsius for about 5 to 10 minutes and then immediately put it on ice for another 5 to 10 minutes.

5. Get the SF1/BBP protein ready by making your protein concentrations 5X the your desired final concentrations. My Kd for this SF1/BBP construct and RNA has been around 1 to 2 uM, so I would recommend your protein concentrations be in the range between 200 nM to 20 uM.

6. Once I have everything ready I mix my samples as follows:
5 uL water
1 uL 10X binding buffer
1 uL RNA-GAGUAAC stem loop
2 uL SF1/BBP protein
1 uL 10X loading dye
------
10 uL Total volume

7. Incubate the binding reaction at room temperature for 5 to 10 minutes and then load the gel which should be at 4 degrees Celsius.

8. Run the gel at 100 volts , 4 degrees Celsius, for about 2 hours.

9. Dry gel, expose and develop.