Western Blots

BB 12/15/04

SLHM 01/07; 07/07

Page 1

Western Blots

Running the Gel

1.  Get Western Blot data sheet & write down all information known before starting

2.  Take gel out of container, remove comb & strip off of the bottom. Choose gel based on size of protein of interest.

3.  Place Running Buffer in top & rinse out wells with bulb pipette

4.  Insert samples with correct micropipette, write down standard number

5.  Fill tank halfway with Running Buffer

6.  Place into tank with protruding edge in the middle of the tank

7.  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!!)

1.  Cut membrane(s) to template gel size

2.  Remove blue covers & place membrane(s) in clear bin

3.  Wash with methanol for 1 min

4.  Quickly rinse with milli Q water

5.  Soak membrane(s) in Transfer Buffer for 15 min on the orbital shaker.

6.  Remove gel from tank

7.  Break gel holder & remove one side (make sure gel pulls off only on one side)

8.  Place a dry blot on the gel to remove it from the plastic holder

9.  Place the membrane directly on the gel

10.  Place a blot wet with Transfer Buffer on the membrane (to make a sandwich)

11.  Dip the dry side in the Transfer Buffer

12.  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

1.  With GLOVES ON, remove the “Sandwich” from the transfer apparatus.

2.  Using tweezers, place the membrane into a container with the block solution.

3.  Place the membrane on the shaker for one hour at Room Temperature.

4.  With gloves still on, place the gel into coomassie blue for staining.

5.  After one hour, begin washing with TBS Tween.

6.  The recommended wash time are as follows

1. 1st -- 15 minutes
2. 2nd, 3rd, & 4th -- 5 minutes

Alternative Methanol Blocking
1.  After removing membrane, place into methanol for 5 minutes.
2.  Dry for 10-15 minutes, then place into primary.
Primary
1.  Make up primary during the block washing.
2.  The Primary antibody is always made in the solution used to block.
3.  Dilute the antibody as directed (if not know, 1:1,000 is always a safe starting point)
4.  Place 20 mL of the primary and the washed membrane in a sealed plastic bag or place 15ml in small container.
5.  Put the bag in the 4ºC room on the rocker for overnight or over the weekend.
6.  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.**
7.  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
1.  The secondary antibody is always the animal that the primary was harvested from.
ie. Primary -- Rabbit Anti-actin
Secondary -- Anti-Rabbit
2.  Make the secondary in the Block solution also
3.  Secondary antibodies are often diluted 1:1,000.
4.  Place the membrane in another Tupperware container and add 20 mL of secondary
5.  Incubation for Secondary is one hour at room temperature on orbital shaker.
6.  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

1. Place a sheet protector into the cassette
2. 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