2/16/2016 RAK TAP protocol revised from Hadley
TAP Purification Protocol
I will use 10 p150 plates of 293T cells for my protein of interest and 4 plates for my vector control. I will continue my new
modifications to the protocol utilizing less IgG bead vol (rec for strepavidin tag 1-5ul beads/mg extracted ptn – no less
than 15ul and no more than 100ul for strep beads – therefore will use 20ul/plate ), using AcTEV Protease at 4 degrees,
markedly increasing the EGTA conc (50mM) in my CEB, and eluting at RT.
Day 1: Transfect Cells
 Transfect each plate with 20ug DNA using FuGENE6 method (1:4 ratio for
293T) Pool each sample into 40ug (2 plate) aliquots
 Make a mix of FuGENE with serum-free media (160ul and 1040ul
respectively per 2 plates) and let rest at RT for 5minutes.
 Add 1200ul diluted FuGENE to 40ul DNA, flick to mix, and let rest at RT
for 15 minutes. Add 620ul mix to each plate dropwise. Incubate for 24
hours before lysis; plates should almost be confluent.
Day 2: Collect Protein Lysates
Make 15ml of NP-40 lysis buffer:150mM NaCl (450ul of 5M stock)
0.1% NP-40 (15ul of 100% stock)
50mM Tris-HCl, pH 8.0 (750ul of 1M stock)
1.5 mini-tabs of Complete Protease Inhibitor
(lyse cells at 60-70% confluence; lyse 7 plates at a time)
 Wash cells 2X with 10ml of cold PBS.
 Add 1ml lysis buffer
 Incubate on ice for 30 minutes, with occasionally rocking
 Scrape and combine 5 plates’ lysates in 15ml conical tubes.
 Quick freeze lysates in lN2 and store in -80.
Day 3: Bind to IgG beads
Make 70 ml IPP150 buffer (add PMSF and Complete mini Tabs)
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Freeze/thaw lysates 3X
Wash 280ul rabbit IgG beads 3X with 3ml IPP150 buffer (20ul/plate)
Spin lysates for 10min at 3,000g, 4ºC.
Remove supernatant and divide 2 plates’ lysates into 15ml conical tubes.
Collect SAMPLE #1 – 40ul of input for protein of interest and vector ctrl.
Add 40 ul (vector) or 50ul (ptn) washed IgG beads and 1:4 vol IPP150.
Rock/rotate overnight at 4ºC.
2/16/2016 RAK TAP protocol revised from Hadley
Day 4: Cleave off IgG beads, bind to calmodulin resin, elute purified ptns
Make 10 ml TEV Cleavage Buffer (add 1ul/ml 1M DTT)
Make 25ml Calmodulin Binding Buffer (add 0.697ul/ml BME and 1ul/ml PMSF)
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Pellet IgG beads, 1800 rpm x 3 minutes.
Collect SAMPLE #2 – 200ul of flow thru.
Combine beads (80ul (4 vector plates) or 100ul (5 ptn plates)) in 3x1.6ml
eppys.
Wash 3X 1ml IPP150
Wash 1x 1ml TEV cleavage buffer
Add 1 ml fresh TEV cleavage buffer and 10ul AcTEV protease
Rotate at 4ºC for 4 hours.
Pellet IgG beads and collect supernatant in 3x2ml eppys
Wash beads with 200ul TEV cleavage buffer and combine supernatants
for a final volume of 1.2ml per eppy.
Collect SAMPLE #3 – 20ul of concentrated protein
Collect IgG beads
Add 3.3ul 2M CaCl2 and 575ul Calmodulin Binding Buffer to each 2ml
eppy.
Wash 140ul calmodulin resin 3X 3ml Calmodulin Binding Buffer
(10ul/plate)
Add 50ul to each ptn of interest eppy and 40ul to vector eppy.
Rock gently for 3-4 hrs at 4C.
Make 1.5ml Calmodulin Elution Buffer (Add 0.697ul/ml of BME)
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Collect SAMPLE #4 – 200ul of flow thru.
Wash resin 3X 1ml Calmodulin Binding Buffer.
Consolidate all like-resins. (100ul ptn of interest, 40ul vector)
Elute protein complexes at RT every 5 minutes using 100ul Calmodulin
Elution Buffer for ptn of interest and 50ul for vector. Flick and occasionally
vortex to help elution.
Combine the 1st and 2nd, 3rd and 4th, and 5th and 6th elutions
Collect calmodulin resin
2/16/2016 RAK TAP protocol revised from Hadley
Day 5: Silver stain to confirm co-purified ptns present, run gel to confirm
ptn of interest present through-out the TAP steps
 Run approximately 12ul of elutions (1&2, 3&4, and 5&6) on a 4-12% NuPAGE gel for silver stain (silver SNAP protocol).
o 12ul sample volume, 2ul 10x reducing agent, 6ul 4x sample buffer
heated to 68c for 10 minutes.
 Use Benchmark diluted 1:100 in dH20 and add the running sample buffer
(33ul of 4X) and then run 10ul vol in gel)
 Dry silver stain gels to save using gel drying kit.
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Run 12ul of saved sample aliquots, IgG beads (boiled in sample buffer),
and calmodulin resin (boiled in sample buffer), along with 12ul of each
construct’s elution on a 4-12% Nu-PAGE gel prepoured for western (when
thawing protein elutions, mix well, and then spin down to assure protein is
suspended).
o 12ul sample volume, 2ul 10x reducing agent, 6ul 4x sample buffer
heated to 68c for 10 minutes.
Transferred at 300mAmps for 4 hours o/n.
Day 6: Western blot to confirm tagged protein of interest expression and
elution
 Blocked with 5% milk/TBS-T for 1 hour
 Added primary antibody for 1 hour.
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Washed 3 times, 10 minutes each, with 1x TBS-T.
Added secondary antibody for 1 hour in 5% milk/TBS-T.
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Washed 4 times, 10 minutes each, with 1x TBS-T.
Added lumilight and exposed to film.
2/16/2016 RAK TAP protocol revised from Hadley
Materials to buy:
IgG Sepharose 6 Fast-Flow (Pharacia 17-0969-01, 10ml)
Calmodulin affinity resin (Stratagene 214303)
AcTEV Protease (Invitrogen, 12575-015, 1000units)
4-12% Nu-PAGE gels (Invitrogen, NP0321BOX)
NuPAGE sample reducing agent (Invitrogen, NP0004)
NuPAGE LDS sample buffer (Invitrogen, NP0007)
NuPAGE MES SDS running buffer (Invitrogen, NP0001)
GelCode SilverSNAP Kit II (Pierce, 24612)
Gel Drying Kit (Invitrogen, N12387)
Buffers to make (All sterile filtered and stored at 4 degrees. CEB stored at RT):
IPP150:
10mM TrisHCl, pH 8.0
10ml 1M stock
150mM NaCl
30ml 5M stock
0.1% NP-40
1ml 100% stock
Bring up to 1000ml in H2O. Add protease inhibitors just before use
TEV cleavage buffer:
10mM Tris-HCl, pH 8.0
10ml 1M stock
150mM NaCl
30ml 5M stock
0.1% NP-40
1ml 100% stock
Bring up to 1000ml in H2O. Add 1ul 1M DTT/ml TEVCB just before use
Calmodulin binding buffer:
10mM Tris-HCl, pH 8.0
10ml 1M stock
150mM NaCl
30ml stock
1mM MgOAc2
1ml 1M stock
1mM imidazole
500ul 2M stock
2mM CaCl2
2ml 1M stock
Bring up to 1000ml in H2O. Add 0.697ul BME/ml CBB and protease
inhibitors just before use
Calmodulin elution buffer:
10mM Tris-HCl, pH 8.0
10ml 1M stock
150mM NaCl
30ml 5M stock
1mM MgOAc2
1ml 1M stock
1mM imidazole
500ul 2M stock
50mM EGTA
4ml 0.5M stock
Bring up to 1000ml in H20. Add 0.697ul BME/ml CEB just before use.