PREPARE LYSATE



Lyse cells for 20-30 min on ice in CHAPS lysis buffer



Spin cells at 4°C for 20 min



Save supernatant in a new RNase-free tube



Quantify protein concentration using the BioRad kit. Be sure to have appropriate blanks and concentration controls



Dilute a small portion of the sample to 50 ng/μL. Return the undiluted sample to -70°C for future use



Take one quarter of the diluted sample and boil for 5 min and crash on ice. This will serve as your heat inactivated control





END LABEL TS OLIGO

(this makes enough oligo for 10 reactions):



Mix:



§ 2.5 μL γ-32P-ATP (3000 Ci/mmol)



§ 2 μL 10X buffer



§ 5.2 μL TS primer



§ 9.8 μL ddH2O



§ 0.5 μL T4 kinase



Heat to 37°C for 20 min



Heat to 85°C for 5 min





SET-UP TRAP REACTION



Set-up the following 50 μL reaction (this is per reaction so when performing multiply reactions prepare a supermix). Each sample should have a minimum of two conditions:
the sample and the heat inactivated control. You can also set up a dilution series if you are attempting to quantify the activity



Mix:



§ 5 μL 10X buffer



§ 2 μL Primer



§ 2 μL labeled TS primer



§ 1 μL dNTPs (at 2.5 mM each)



§ 0.4 μL Taq



§ 37.6 μL ddH2O



§ 2 μL prepared extract





RUN REACTION



Set up PCR program:



1 cycle



30 min at 30°C



27 cycles



94°C for 30 sec

60°C for 30 sec





RUN GEL



12.5% acrylamide gel (0.4mm) with 0.5X TBE buffer



Add 10 μL of 6X loading dye and run 6 μL



Run gel at about 1000-1200 volts until the first dye runs off and the second dye is about 3/4 the way into the gel





IMPORTANT BUFFERS:



1X CHAPS (3-[(Cholamidopropyl)dimethylammonio]-1-propanesulfonate) buffer (All Rnase-free)



Mix:



¨ 10 mM Tris, pH 7.5



¨ 1 mM MgCl2



¨ 1 mM EGTA



¨ 0.1 mM benzamidine



¨ 5 mM 2-mercaptoethanol



¨ 0.5% CHAPS



¨ 10% glycerol





Immediately before lysing cells add 5 mM -mercaptoethanol (stock from Sigma is at 14.3 M so add 0.5 μL to 1.4 mL of the buffer)



10X TRAP BUFFER



Mix:



¨ 200 mM Tris, pH 8.3



¨ 15 mM MgCl2



¨ 630 mM KCl



¨ 0.5% Tween 20



¨ 10 mM EGTA



¨ 0.1% BSA





50X dNTPs



2.5 mM of each dNTP





OLIGOS:
(Oligos ordered purified in solution)





ACX: GCGCGGCTTACCCTTACCCTTACCCTAACC



TSNT: AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT



NT: ATCGCTTCTCGGCCTTTT



TS: AATCCGTCGAGCAGAGTT









CONCENTRATIONS FOR USE:



Primer mix II:



§ ACX 0.1 μg/sample



§ NT 0.01 μg/sample



§ TSNT 0.01 amol/sample





Primer mix III:



§ ACX 0.1 μg/sample



§ NT 0.1 ng/sample



§ TSNT 0.01 amol/sample