Sfeir Lab, Aleks Penev, 8/21/14

Western Blot
Sample Prep & Loading
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Trypsinize, collect and count 500K using Cell Counter.

Spin down @ 1200 RPM to pellet cells.
Wash and re-spin 2x with PBS.
Resuspend pellet in 25 ul PBS.
Add 25 ul 4x LB (from -20 common). Works best if hot, so boil LB before adding.
Triturate sample several (5-10) times.
Boil 5 minutes at 95 (sand) and then cool on ice.
Assemble BioRad Tetrapack gel running apparatus with plastic dam if running an odd
number of gels. Fill to appropriate mark on side and fill gel cassette to the top with
running buffer.
9. Load 10ul of protein ladder (BioRad, -20C ladder box).
10. Load up to 40ul based on desired strength of signal. Usually 25ul is sufficient.
11. Run gels at 90V for 1:40hrs (100min). This is usually enough for the gel front to reach
the end of the gel. You may choose to run longer based on the product in question.
*You can also run gel at 20V O/N in cold room but this is not recommended.
Transfer
1. Ensure that transfer buffer has chilled to 4C before using. Buffer may be reused once.
2. Cut one piece Protran nitrocellulose and 4 pieces of Whatman paper to roughly the size of
the gel without the stacking layer.
3. Arrange transfer cassette in shallow dish with transfer buffer and roller to smooth out
bubbles: place nitrocellulose on gel, then place 2 whatman pieces on either side. Then
place one sponge on either side of stack and clip into place in transfer cassette.
4. Arrange transfer cassettes in the proper orientation (membrane towards red side and gel
on black side) and transfer in the cold room. Either 90V for 1:30hrs or 25V O/N.
Blotting
1. Immediately after transfer finishes, begin blotting membrane in 5% milk (aka Blotting
Grade Blocker) in TBST. Incubate at RT on shaker for 1 hr.
2. Dilute primary antibodies in blocking solution (5% milk in TBST):
-Myc 9E10
use 1:5000
(4 common)
-GT488 (-tubulin) use 1:10,000 (-20 common)
Only 5 ml necessary per membrane.
3. Place membrane and primary antibody in plastic bag and seal using the heat sealer,
avoiding bubbles in the bag.
4. Incubate at RT on rotator for 2 hrs.
5. Wash 3x 5min in 1x TBST on rotator.
6. Begin to prepare 5ml of diluted secondary antibody in blocking solution:

Mouse-HRP use 1:5000

(4 common, small black box)
7. Place secondary antibody solution in bag with membrane, seal in bag and incubate at RT
on rotator for 45 min.
8. Wash 3x 5min in 1x TBST.
9. Rinse in 1x TBS (no Tween) to reduce background.
Developing
1. Mix 750ul of each Amersham ECL reagent, enough to cover the membrane. Incubate 1-2
minutes.
2. Drain developing reagent off, dab excess with a Kimwipe and place in cassette with
plastic liner.
3. Expose grey film in darkroom for 1, 5, and 10 minutes. Several-hour or O/N exposure is
possible for weak signals, but most signal is recovered in the first few hours.
*If signal is weak, use PicoPure ECL reagent instead: incubate 2 minutes and expose
minimum 1 hour.
4. Once done developing, make sure to align film to membrane in cassette before anything
else to mark ladder on film in order to properly interpret the results. Use fluorescent
markings inside the cassette to properly align film inside cassette.