Yu Lab 2/26/2012

1

STAT5 DNA BINDING ASSAY
OLIGONUCLEOTIDE PULL-DOWN

I. Cell Pellet Lysis
1. Prepare 0.5% NP40 Cell Lysis Buffer.
add PMSF and TLA before use
2. Add ice cold cell lysis buffer into sample tube and mix by pipette gently.
buffer volume depend on cell pellet size.
3. Rotate at 4C for 60~120 min.
4. Spin down at top speed for 30 min at 4C.
5. Transfer supernatant to a new tube
6. Check protein concentration by Bio-Rad reagent or BCA protein assay.

II. Oligonucleotide Pull-Down
1. Normalize whole cell lysates to same amount of total proteins and volume of lysates.
Keep lysates cold on ice. (Discuss with Dr. Yu)
2. Transfer lysate into beads and keep cold. Mix well by invert.
Get DNA pre-treatment streptavidin-sepharose beads form Dr. Yu
3. Put microfuge tubes on the rotator at 4C, O/N.
4. Quick spin down ~10,000 rpm at 4C.
5. Let beads settle for 2 min on ice, carefully remove supernatant with vacuum suction.
6. Add 1 ml of cold IP wash buffer into each tube.
7. Thoroughly resuspend sepharose by inverting the tubes.
8. Quick spin down ~10,000 rpm at 4C.
9. Let beads settle for 2 min on ice, then carefully remove wash buffer with vacuum suction.
10. Repeat the IP wash buffer wash two more times. (Total 3 times)
11. Make final wash with 1 ml of ice cold 1X PBS.
12. Let beads settle for 5 min, then carefully suction off.
13. Add 30~35l 1X sample loading buffer (+ 5% 2MP )/ tube
For 35l: 5.8l 6X sample loading buffer + 1.8l 2MP + 27.4l ddH
2
O
14. Boil (100C) the sepharose beads for 5 minutes.
Yu Lab 2/26/2012
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15. Spin at maximum speed in microfuge for 1 minute. Can store at -20C or loading to the
western gel.
16. If samples had been store at -20C, need re-boil the sepharose beads for 5 minutes and
Spin at maximum speed in microfuge for 1 minute befor loading to the western gel.
17. Load the 35 l supernatant in the gel. (8% SDS gel)

III. Western Blotting
1. Wash the membrane 4 times by 1XTBST, ~5 min/time
2. Block the non-specific binding sites by incubating the membrane in 5% non-fat
milk in 1XTBST (0.5g milk + 10 ml) for 1-2 hour at RT.
3. Wash the membrane 6 times by 1XTBST, ~5 min/time (or 4 times, 10min/time)
4. Primary Antibody
Blotting membrane with anti-STAT5 antibody (SC835, rabbit) for O/N at 4C.
20l antibody+ 8ml 1XTBST (0.5g/ml)
5. Wash the membrane 6 times by 1XTBST, ~5 min/time
6. Enhanced chemiluminescence (ECL):
Dilute 1.6l IgG rabbit HRP (SCBT, SC2004) in 8 ml 1XTBST.
For 1 hour at RT.
7. Wash 6 times in 1XTBST for 5 min/time (or 4 times, 10min/time).
8. Add ~1 ml Western HRP Substrate (Millipore), for 2~5 min at RT.
9. To drain excess reagent, hold it vertically and touch it against tissue paper.
10. Place the membrane between the covers of a polypropylene sheet protector with the
interleaf removed. Gently smooth out any air pockets.
11. Place the membrane, protein side up, in the film cassette.
12. Dark room. Take image.


Yu Lab 2/26/2012
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IP Wash Buffer:

Total Vol. 100 250
Stock Conc. Final Conc. ml ml
1 dd. H2O 89.6 224
2 Tris (pH8.0) 1 M 50 mM 5 12.5
3 NaCl 5 M 150 mM 3 7.5
4 Na3VO4 0.1 M 1 mM 1 2.5
5 EDTA 0.5 M 1 mM 0.2 0.5
6 EGTA 0.5 M 1 mM 0.2 0.5
7 Triton X-100 (or NP40) 10% 0.1% 1 2.5



0.5% NP40 Cell Lysis Buffer

Total Vol. 1000 1500 2000 3000 4000 5000
Stock Conc. Final Conc. ul ul ul ul ul ul
1 dd. H2O 716 1074 1432 2148 2864 3580
2 Tris (pH8.0) 1 M 50 mM 50 75 100 150 200 250
3 NaCl 5 M 150 mM 30 45 60 90 120 150
4 Na3VO4 0.1 M 1 mM 10 15 20 30 40 50
5 NaF 0.5 M 10 mM 20 30 40 60 80 100
6 Na4P2O7 100 mM 10 mM 100 150 200 300 400 500
7 EDTA 0.5 M 1 mM 2 3 4 6 8 10
8 EGTA 0.5 M 1 mM 2 3 4 6 8 10
9 NP-40 10% 0.5% 50 75 100 150 200 250
10 TLA (-20C) 100 X 1 X 10 15 20 30 40 50
11 PMSF (-20C) 200 mM 2 mM 10 15 20 30 40 50

Total loading Vol. 1X 5%
l (l) 6X loading buffer 98% 2MP ddH2O
1 600 100.0 30.6 469.4
2 510 85.0 26.0 399.0
3 450 75.0 23.0 352.0
4 360 60.0 18.4 281.6
5 240 40.0 12.2 187.8
6 150 25.0 7.7 117.3
7 35 5.8 1.8 27.4
15 30 5.0 1.5 23.5