WESTERN BLOT
I. SDS-PAGE
1. Precast polyacrylamide gels have been purchased for use in this lab. Polyacrylamide is a
neurotoxin and most harmful to human health in powder form. Wear gloves and use all safety
precautions when handling PAGE precast gels.
2. Your group will be receiving either the gradient (10-20%) or the fix (10%) gel. Cut open the
pouch and remove the gel. THE GEL IS EXTREMELY FRAGILE-USE THE UTMOST
CARE WHEN HANDLING THE PRODUCT.
3. Rinse the cassette with distilled water.
4. Peel the tape off the bottom of the cassette.
5. Gently pull the comb out of the cassette BE VERY CAREFUL NOT TO RIP GEL. Put the
comb aside so it can be used later to separate the cassette plates.
6. Mount the cassette into the electrophoresis apparatus so the printed side faces the outer (anode)
buffer chamber.
7. Fill the buffer chambers with the appropriate amounts of running buffer. Use approximately
150ml in the anode chamber and 250ml in the cathode chamber. Your TA will confirm these
volumes during the lab session
8. Use a pipette to wash the sample wells with 1X running buffer, displacing any air bubbles in
the wells. Load 20ul of the following samples into each well.
SAMPLE WELL
SAMPLE
1
Pre-stained standard proteins
2
Cow serum
3
Horse serum
4
Goat serum
5
Pre-stained standard proteins
6
Sheep serum
7
Donkey serum
8
Chicken serum
The TA will load the first lane so that you can see the lanes. The TA will have suggestions on
how better to see the lanes for loading.
9. Attach the electrophoresis apparatus to the power supply and run the Tris-Glycine gels at a
constant voltage of 200V until the dye front is near the bottom of the gel. The TA will execute
this part of the experiment.
10. When the run is complete, shut off the power and remove the gels. The TA will help in the
disassembly of the gel.
11. Place the cassette on a flat surface with the notched (well side) facing up. Starting near the top,
work your way around the cassette using the end of the comb to separate the two plates.
Carefully remove the short plate. The gel will adhere to either the short or long plate. Mark
orientation using a razor blade by sniping off corner to indicate well no 1.
12. Hold the plate with the gel over an open container containing either CAPS buffer or gel stain
(this will depend on group assignment). If the gel is adhered to the larger plate carefully insert a
flat edged device (comb) through the plate’s slot and gently push out the bottom of the gel;
allow the gel to peel away and gently drop into the container. If the gel is adhered to the smaller
plate carefully use the comb to loosen one lower corner of the gel; allow the gel to peel away
and gently drop into the container.
II. Transferring to Membrane
During the rest of the experiment it is important that clean gloves be worn when handling all
material. Therefore, rinse gloves with distilled water before handling gels and nitrocellulose
paper. Take care not to touch anything once you have rinsed the gloves.
13. Take nitrocellulose and mark (with a pencil) in the upper left hand corner so that the correct
orientation will be kept throughout the experiment.
14. In a clean plastic container wet blotting paper and nitrocellulose in ~30ml of 20mM CAPS
buffer pH=10. Hydrate nitrocellulose membrane by slowly sliding it into the buffer at a 45
angle.
15. Abrupt wetting may trap air bubbles in membrane and lower efficiency of transfer. Let soak for
10-15 minutes with shaking.
16. Construct blot sandwich as follows:
Note: The orientation of the stacking is important. Proteins will move from the Cathode
(bottom plate) to the Anode (top plate).
2 sheets of blotting paper (roll with test tube after each to release air bubbles)
Polyacrylamide gel
Nitrocellulose (Nitrocellulose should be toward the anode)
2 sheets of blotting paper
Turn power supply on to 10V and transfer for 30 minutes. THE SEMI DRY TRANSFER
CELL IS A HIGH CONDUCTOR OF ELECTRICITY. DO NOT TOUCH THE UNTI WITHOUT THE
INSTRUCTORS CONSENT.
17. Turn off and disconnect power supply. Rinse gel and blot in separate containers with distilled
water. Stain the gel with Commassie blue stain as previously performed.
18. PROTEIN STAINING OF THE NITROCELLULOSE MEMBRANE
1 group will stain the blot with Ponceau S by adding 15ml of Ponceau S stain to blot for 5
minutes. Stop reaction by rinsing membrane with distilled water. This stain will show the total
amount of protein transferred to the nitrocellulose membrane.
19. IMMUNOLOGICAL DETECTION PROCEDURE FOR THE NITROCELLOSE
MEMBRANE
3 groups will conduct the immunological procedure. Take the membrane and incubate in 15ml
of gelatin solution (WHY?) for 15 minutes on the shaker.
20. ANTIBODY REACTION
A. Dilute the antibody 1:100 by adding 50ul of antibody to 5ml of fresh gelatin solution in a
petri dish. Transfer the blot to the dish and gently swirl to ensure that all surfaces of the blot
are exposed to the antibody solution.
B. Place lid on dish and float in water bath at 37C for 25 minutes.
C. Transfer container to plastic containers and wash for 3-5 minutes in 20ml of the following
solutions. Manual rocking or shaking should be performed during these washes.
TBS+NP40 x 4
TBS
21. DETECTION OF ANTIBODY
A. Instructor will prepare Color Development Solution as described in lab manual.
B. Pour off last wash and replace with Color Development Solution. Gently rock for ~10
minutes. Rinse the blot in water and examine both sides.