2/17/2016 RAK
TRAP assay (TRAPeze Telomerase Detection Kit, Chemicon #S7700)
Collect cells for TRAP assay
We typically do TRAP assays on human foreskin keratinocytes (HFKs). 1/3 of a confluent 10cm 2 plate yields 300ug of protein for
this assay.
1. Cells are typsinized in the usual fashion and pelleted in a 15ml conical tube.
2. Cells are washed in PBS and spun at 4°C (4-14,000rpm for 10 minutes).
3. PBS is removed and the dry pellet of cells is snap frozen in liquid nitrogen and stored at 80°C until needed.
Lyse cells for protein extraction
1. Thaw dry cell pellet on ice
2. Add 50-60 ul of CHAPS lysis buffer and incubate on ice of 30 minutes
3. Spin down lysates at 12,000xg (11,000 rpm) for 20 minutes at 4C
4. Remove supernatant and place in a fresh eppy
5. Calculate protein concentration using the Biorad Assay. Typically for cellular extracts,
use 2ug and 0.5ug per assay. Based on your experiment, less protein can be used for a
reaction. (We have used 0.1ug of protein per reaction with good results.)
6. Make the protein concentration 1ug/ul for each lysate, using additional CHAPS lysis
buffer if needed.
7. Aliquot and quick freeze the remaining extract and store at -80C. The extract can keep
for at least 12 months at this temperature.
Biorad Assay for protein concentration
1. Thaw 10mg/ml BSA stock.
2. Aliquot into 1.7ml tubes for the protein assay-BSA: 0, 5, 10, 20, 40, 80, and 120ug (0.512ul of 10mg/ml stock). Also aliquot 5-20ul of each lysate to be tested based on the size
of your cell pellet – if your protein concentration is likely dilute, utilize more volume to
assure it falls within the std curve of the assay.
3. Make mix of reagents A + S (980ul A: 20ul S).
4. Aliquot 125ul of the mix to each tube, vortexing after adding.
5. Add 1ml of reagent B to each tube, vortexing after adding.
6. Incubate at RT for 15 minutes.
7. Aliquot 125ul of each reaction into a 96 well plate for reading in the microquant plate
reader. (Use JAB-protein protocol in KC Junior software).
8. Read plate and print data. Divide the calculated concentration by the ul volume of
protein tested to get the correct concentration.
9. Calculate necessary volume that equals 2 ug of total protein for each lysate sample.
End-labeling of TS primer (T4 Kinase and buffer, Invitrogen #18004-010)
1x
(for 20 assays)
32P-ATP (3000 Ci/mmol, 10mCi/ml)
0.1
2 ul (0.015mCi)
TS Primer
1
20 ul
5X Kinase Exchange Buffer
0.4
8 ul
T4 Polynucleotide Kinase
0.1
2 ul (10U/ul)
dH20
0.4
8 ul
Total
2 ul 40 ul
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Incubate at 37C for at least 20 minutes, then heat inactivate the kinase at 85C for 5
minutes
Use 2 ul per TRAP reaction, purification not recommended
2/17/2016 RAK
Set-up TRAP reactions (Taq polymerase, Invitrogen #18038-042)
1X
(for 20 assays)
10X TRAP reaction buffer 5 ul
100 ul
50X dNTP mix
1 ul
20 ul
32
P-TS primer
2 ul
40 ul
TRAP Primer mix
1 ul
20 ul
Taq Pol (5U/ul)
0.4ul (2U)
8 ul
dH20
38.6 ul
772 ul
Total
48 ul
960 ul
Notes:
 Aliquot 50X dNTPs into 20 uls and replace in freezer (avoids multiple freeze/thaws)
 Thaw all reagents and store and mix on ice



Aliquot 48 ul per tube/sample
Add protein lysates to each tube, 2 reactions for each sample: 2ug and 0.5ug. Dilute down
the protein 4-fold to make the 0.5ug samples in 2ul. (If TRAP activity will be high
consider 1ug, 0.5ug, and 0.1ug)
PCR positive control is TSR8; Telomerase positive control is the cell pellet given
(typically HeLa cells). Use 1ul of each. (Cell pellet for positive control – lyse in 100ul
CHAPS and then dilute it 1:20. Use only 1ul of the dilution)
Cell Lysates
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
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Place tubes in thermocycler (PE 9600)
Incubate at 30C for 30 minutes
2-step PCR: (27 cycles)
94C for 30 sec
59C for 30 sec
Freeze the PCR products
PAGE and Data Analysis
Add 5ul of loading dye (0.25% each BPB and XyC in 50% glycerol/50mM EDTA)
Load and run 25ul on a 10% non-denaturing gel in 0.5x TBE
Run gel 2.5 hours at 250V for 10-12cm vertical gel, or until the XyC runs 70-75% gel and BPB
just runs off the gel
Dry gel and expose to film at -80°C (Biomax MR, Kodak). Quantitate intensity of laddering
seen using ImageJ or Quantity One software.
10% non-denaturing polyacrylamide gel
40% polyacrylamide solution (19:1)
5X TBE
dH20
10% APS
TEMED
50ml
12.5ml
5ml
32ml
500ul
50ul