BB 12/15/04
SLHM 01/07; 07/07
Page 1
Western Blots
Running the Gel
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Get Western Blot data sheet & write down all information known before starting
Take gel out of container, remove comb & strip off of the bottom. Choose gel based on size
of protein of interest.
Place Running Buffer in top & rinse out wells with bulb pipette
Insert samples with correct micropipette, write down standard number
Fill tank halfway with Running Buffer
Place into tank with protruding edge in the middle of the tank
Run the gel for 110 min @ 110 V or sufficient time for protein of interest. Check
molecular weight standard to ensure protein of interest is 2/3 of the way thru the gel before
stopping.
Membrane Transfer (WEAR GLOVES!!)
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Cut membrane(s) to template gel size
Remove blue covers & place membrane(s) in clear bin
Wash with methanol for 1 min
Quickly rinse with milli Q water
Soak membrane(s) in Transfer Buffer for 15 min on the orbital shaker.
Remove gel from tank
Break gel holder & remove one side (make sure gel pulls off only on one side)
Place a dry blot on the gel to remove it from the plastic holder
Place the membrane directly on the gel
Place a blot wet with Transfer Buffer on the membrane (to make a sandwich)
Dip the dry side in the Transfer Buffer
Place the entire “sandwich” into the transfer apparatus MEMBRANE SIDE DOWN
Sandwich going from bottom to top: blot, membrane, gel, blot
13. Roll out air bubbles with an empty conical tube
14. Close the apparatus and run 40 min @ 18 V for two gels (one gel 20 min @ 18V); for 10%
or higher gels , transfer for 60 min @ 12V
Block
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With GLOVES ON, remove the “Sandwich” from the transfer apparatus.
Using tweezers, place the membrane into a container with the block solution.
Place the membrane on the shaker for one hour at Room Temperature.
With gloves still on, place the gel into coomassie blue for staining.
After one hour, begin washing with TBS Tween.
The recommended wash time are as follows
a. 1st -- 15 minutes
b. 2nd, 3rd, & 4th -- 5 minutes
c.
Alternative Methanol Blocking
1. After removing membrane, place into methanol for 5 minutes.
2. Dry for 10-15 minutes, then place into primary.
BB 12/15/04
SLHM 01/07; 07/07
Page 2
Primary
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Make up primary during the block washing.
The Primary antibody is always made in the solution used to block.
Dilute the antibody as directed (if not know, 1:1,000 is always a safe starting point)
Place 20 mL of the primary and the washed membrane in a sealed plastic bag or place 15ml
in small container.
Put the bag in the 4ºC room on the rocker for overnight or over the weekend.
Before washing, collect the Primary antibody in a new tube and place it into the freezer.
**Note: Because primary antibodies are so expensive, we save the antibody in block to be
used again. Mark date, antibody info, and how many times used on antibody container.**
Wash the excess primary off of the membrane with TBS Tween in the same manner the
Block was washed. ** 3x 30 seconds wash with TBS-Tween if use methanol.
Secondary
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The secondary antibody is always the animal that the primary was harvested from.
ie. Primary -- Rabbit Anti-actin
Secondary -- Anti-Rabbit
Make the secondary in the Block solution also
Secondary antibodies are often diluted 1:1,000.
Place the membrane in another Tupperware container and add 20 mL of secondary
Incubation for Secondary is one hour at room temperature on orbital shaker.
Wash the excess secondary off of the membrane as previously done. 3X 5 minutes each.
Developing
1.
First, set up a cassette for developing the membrane
a. Place a sheet protector into the cassette
b. Make sure to tape it to the cassette so it does not move around
2. In the fridge find the Western Lighting Chemoluminsence kit.
3. In a new Tupperware container, place equal volumes of the brown bottle liquid & the Clear
Bottle liquid.
4. Remove the membrane from the wash with tweezers and blot between two large pieces of
extra thick blotting paper.
5. Then, place the semi-dry membrane into the Developing solution.
6. Make sure the membrane is saturated with the solution.
7. Place it into the cassette in between the sheet protectors, colors on right side top of gel
shows highest MW.
8. Mash out all bubbles over the membrane with the back side of a razor blade.
9. Take multiple exposures of the membrane while in the dark room.
10. Make sure to label the films BEFORE you remove the membrane from the cassette.
11. The membrane can be stored in regular TBS for future use.
Recipes
Running Buffer: 10ml 10X Running buffer (BioRad) and 990 ml H2O
Transfer Buffer: 10ml 10X Transfer buffer (BioRad) and 990 ml H2O
TBS-TWEEN: 500ml Tween, 10ml 10X TBS, 985ml H2O