Ni-NTA Column, His-tag Protein Purification
 Sonicator needs to “warm up.” So turn on and add ice to bring temp. down.
1) Resuspend cells in 800 uL ice cold Lysis Buffer (per 20 million cells).
2) Consolidate into large eppie.
3) Leave samples on ice for 15 min. Sonicate (in ice, ~0°C) for 5 min.
- Press Set Sonics, then ON button. Keep own timer for 5 min.
4) Balance eppies. Centrifuge at 12,000 rpm in cold room (4°C) for 18 min.
5) Transfer supernatant to new tube. Balance again and repeat step 5.
 While centrifuging, equilibrate 2 mL Ni-NTA beads (0.5 mL per column) with
40 mL Wash Buffer (10 mL per 0.5 mL beads).
 Centrifuge beads down (in Cell Culture Facility) at 1000 rpm at 5°C for <5
min. Pipet off Wash Buffer.
6) Remove 10 uL from supernatant (clarified lysate) and store for SDS-PAGE.
7) Take rest of supernatant and add to Ni-NTA beads (equilibrated in Wash Buffer).
Rotate samples in cold room for 1 h.
8) Clamp columns to ring stands. Rinse hoses w/ddH2O, and then run ~10 mL Wash
Buffer through.
9) Cut piece of hose and attach to end of column. Clamp. Mix beads/supernatant gently,
then pipet onto column. Let beads settle.
10) Let supernatant run through ‘til small head volume above column (should try to
maintain this throughout purification).
11) Either attach hoses to buffers and run through columns or pipet buffers on top of
columns manually.
12) Unclamp column and run ~20 mL Wash Buffer through column. Collect flow-thru in
50-mL conical.
13) Prepare 96-well plate with 100-uL Bradford reagent per well.
14) Add Elution Buffer w/glass pipet. Collect ~500 uL fractions in large eppies. Add 10
uL of each 100-uL Bradford reagent.
15) Collect fractions that turn Bradford reagent blue (indicates protein present). Rinse
dialysis bag w/ddH2O and clip one end w/orange clips.
16) Save ~50 uL of protein in case of mistakes during dialysis. Take rest of protein and
add to dialysis bag. Clip other end and place in Dialysis Buffer. Make sure stir bar
spinning at rate fast enough that dialysis bag is slowly rotating in buffer. Dialyze
overnight.
17) …. Following morning, unclip dialysis bag and transfer entire volume to 1 or 2 large
eppies. Pipet 10 uL aliquots into small eppies and store in freezer box(es). Label cover
(and eppies if necessary). Store at -80°C.
Lysis buffer
Nonidet-P40
NaCl
Tris, pH 8
glycerol
MgCl2
-mercaptoethanol
Begin. Conc.
20 %
End Conc.
0.1 %
400 mM
20 mM
5%
1.5 mM
8 mM
Vol. in 10 mL
50 uL
0.234 g
0.2 mL
625 uL
15 uL
5.6 uL (add just
before using)
1 tablet (add just
before using)
Adjust to 10 mL
1M
80 %
1M
14.3 M
End Conc.
0.1 %
400 mM
20 mM
5%
1.5 mM
8 mM
2M
28 mM
Vol. in 250 mL
1.25 mL
5.8 g
5 mL
15.6 mL
375 uL
139.9 uL (add just
before using)
3.5 mL
Adjust to 250 mL
Begin. Conc.
20 %
1M
80 %
1M
14.3 M
End Conc.
0.01 %
400 mM
20 mM
5%
1.5 mM
8 mM
2M
200 mM
Begin. Conc.
End Conc.
10 mM
20 mM
10 %
1.5 mM
1mM
1M
80 %
1M
14.3 M
Complete Mini EDTA-free tablet (Roche
diagnostics; protease inhibitor cocktail)
Milli Q water
Wash buffer
Nonidet-P40
NaCl
Tris, pH 8
glycerol
MgCl2
-mercaptoethanol
Imidazole
Milli Q water
Begin. Conc.
20 %
Elution buffer (200 mM imidazole)
Nonidet-P40
NaCl
Tris, pH 8
glycerol
MgCl2
-mercaptoethanol
Imidazole
Milli Q water
Vol. in 20 mL
10 uL
0.468 g
0.4 mL
1.25 mL
30 uL
11.2 uL (add just
before using)
2 mL
Adjust to 20 mL
Dialysis buffer
NaCl
Tris, pH 8
glycerol
MgCl2
DTT
Milli Q water
1M
100 %
1M
Vol. in 4 L
2.34 g
80 mL
400 mL
6 mL
1.14 g
Adjust to 4 L
SDS-PAGE and Western Blot
1) Clean 4 plates (2 large, 2 small) with ddH2O and ethanol. Place (thinner) spacers
between plates and tighten into gel holders with black screws. Make sure spacers flush
with bottom. Snap gel holders onto foam of gel-pouring apparatus.
2) Make Separating (7%) Gel Solution:
2.75 mL
2.8 mL
6 mL
113 uL
56 uL
7.6 uL
30% Acrylamide
1.5 M TRIS, pH 8.8
ddH2O
10% SDS
10% APS (make fresh!)
TEMED
3) Using a Pasteur pipet, pipet Separating Gel Soln in between plates ‘til just above mark.
4) Add ddH2O on top to give nice straight edge to gel. Let polymerize for 20-30 min.
5) Make Stacking Gel Solution:
0.52 mL
1.1 mL
2.8 mL
45 uL
22.5 uL
4.5 uL
30% Acrylamide
0.5 M TRIS, pH 6.8
ddH2O
10% SDS
10% APS
TEMED
6) Remove ddH2O from top of gels. Using a Pasteur pipet, pipet Stacking Gel Soln to top
of plate. Place comb in. Let polymerize for 30+ min.
7) During wait, make up GLB (Gel Loading Buffer) and samples. GLB is 1 part 1 M
DTT and 9 parts 3X SDS Sample Buffer (Red Soln).
8) Use 3 uL of sample (depending on how concentrated it is), 7 uL ddH2O, and 5 uL
GLB. Heat samples at 90°C for 3-5 min.
9) Make Running Buffer (1 L) of 100 mL 10X TRIS/Glycine/SDS buffer and 900 mL
ddH2O.
10) Place gels in gel holder with horns fitting through holes. Put this into the gel box.
Add Running buffer into middle and then outside of gels. Remove combs.
11) Load 10 uL of each sample into wells. Load 5 uL of Protein Ladder.
12) Snap green lid on top. Set to 200 Volts (at constant voltage) and run for 30-40 min.
(or until red dye runs off). If staining with Coomassie, go to Section A; if running a
Western blot, go to Section B.
A. Staining with Coomassie.
13) Remove gels from plates and place in Coomassie Blue soln. Microwave for less
than 1 min. Let rotate for 5-10 min.
14) Pour off Coomassie and SAVE. ADD ddH2O FIRST AND RINSE!! Then add
Destain Solution (45% Methanol, 45% ddH2O, 10% Glacial Acetic Acid). Microwave
for less than 1 min. Let rotate for several minutes. Use kimwipes to soak up excess
Coomassie soln. (Can remove Destain soln, add fresh soln and repeat in microwave.)
15) If necessary, can leave gel in ddH2O overnight. Otherwise, dry gel.
16) To dry gel, make sure trap is full of dry ice and ethanol. Turn pump on. Turn knob
towards “D” for gel dryer.
17) Wet filter paper (cut larger than gel) and lay on dryer. Lay gel(s) on top of filter
paper. Lay cellophane on top of gel (cut larger than gel).
18) Lay cover film on top of gel sandwich. Set temp. at 80°C and time for 45-50 min.
19) Remove cover film and let gel dryer cool down.
B. Running a Western blot
13) Alternately … prepare 1 L of Transfer Buffer, pH 8.3 (100 mL of 10X
TRIS/Glycine/SDS buffer, 150 mL Methanol, and 750 mL ddH2O):
(Final Concentrations)
15% Methanol
25 mM TRIS
192 mM Glycine
0.1% (w/v) SDS
14) Make Western blot “sandwich”:
On black square (w/holes), place:
a. one Scotchbrite pad (soaked in transfer buffer)
b. three Whatman 3 MM pieces (soaked in transfer buffer)
c. gel (DO NOT let gel dry out!!)
d. nitrocellulose (soaked in ddH2O, then transfer buffer)
- cut corner to know orientation
e. three Whatman 3 MM pieces (soaked in transfer buffer)
- roll gently w/glass tube to remove air bubbles
f. one Scotchbrite pad (soaked in transfer buffer)
15) Fold and clamp white square (w/holes) piece to black piece. Place in electrophoresis
apparatus w/black piece facing black side of apparatus.
16) Fill container w/transfer buffer. Run Western @ 150 milliamps for 1 hr. @ room
temp.
17) Make 5% Milk Blocking Solution (2.5 g powdered milk in 4°C 50-mL 1X PBS).
18) Soak Western blot membrane in Blocking solution overnight (O/N) @ 4°C on rotator
below Goodrich bench in cold room.
19) The following morning … Wash w/50 mL PBS-Tween (1X PBS, 0.1% Tween) once
for 15 min, then twice for 5 min. each.
1 uL Tween per 1 mL 1X PBS
20) Incubate in 1:1000 dilution of primary antibody in PBS-Tween (UL5/UL52/UL8
antibodies) for 1 hr.
- SAVE solution, store @ -20°C
21) Wash w/50 mL PBS-Tween three times for 15 min. each.
22) Incubate in 1:2500 dilution of secondary antibody in PBS-Tween for 1 hr. (usually
1:5000 if secondary antibody is newer). Antibody is Goat Anti-Rabbit IgG (H+L) HRP
Conjugated (Chemicon Cat. #AP307P).
- make solution fresh each time
23) Wash w/50 mL PBS-Tween three times for 15 min. each.
Detection (w/help from Michelle Emrick, Ahn lab):
24) Place nitrocellulose membrane on piece of saran wrap. Mix 1.5 mL each of ECL
Detection Reagents, then drip on membrane until entire surface covered.
25) Let sit for 1 min. (DO NOT EXCEED TIME!) Tilt membrane to drain off solution.
Wrap w/saran wrap (avoid air bubbles) and stick on glow-in-the-dark stickers for
orientation. Keep in x-ray film cassette.
26) In dark room:
a. Turn off lights.
b. Take out sheet of film.
c. Place on top of membrane.
d. Time for various amounts (15 sec, 30 sec, 1 min.)
e. Remove film and place into developer machine.
f. Red light will turn off. When red light comes back on, room lights can be
turned back on.