Granato Laboraoty
Laura Gordon
Modified 2/9/2009
Western Blot Protocol
REAGENTS
Separating Gel 10%
Distilled H2O
4.44ml
1.29M Tris-HCl pH 8.8
2.91ml
10% SDS stock
100ul
Acrylamide/Bis 40%
2.5ml
10% APS*
50ul
Temed
6ul
TOTAL
10ml
*To make 10% APS add 100mg APS in 1ml H2O
Stacking Gel 4%
Distilled H20
0.5M Tris-HCl pH 6.8
10% SDS
Acrylimide/Bis 40%
10% APS
Temed
TOTAL
6.1ml
2.626ml
100ul
1.3ml
50ul
10ul
10ml
1X Running Buffer (500mls)
50ml 10X Tris/glycine
0.5g SDS
add H2O to 500mls
1X Transfer Buffer (1 Liter)
100ml 10X Tris/glycine
200ml Methanol
add H2O to 1 Liter
10X Tris/glycine
30g Tris
144g Glycine
Add H20 to 1L
2X SDS Sample Buffer
0.1M Tris-HCl pH 6.8
20% Glycerol
4% SDS
Small pinch of bromophenol blue
Add 5% BME fresh before use
TBS/Tween (500ml)
50ml 10X TBS
0.1% Tween 20
Stripping Buffer
5ml 10% SDS
3.125ml 0.5M Tris pH 6.8
H2O to 25ml
200ul B-mercaptoethanol
PROCEDURE
1. Insert the comb into the secured gel apparatus and draw a line approximately 1’’
below the edge of the comb
2. Make separating gel by adding ingredients in order listed and mix well in a 50ml
conical tube. Add to space between glasses 1ml at a time, adding from the middle
rather than one of the sides until liquid is loaded to the line. Next, add 1ml of water to
the top of the gel to get rid of air bubbles and help catalyze the solidifying reaction.
Let the gel harden for 20 minutes
3. After the separating gel has hardened, pour of the water and wick remainder with a
kim-wipe. Make the stacking gel and add to the top of the separating gel. Let harden
an additional 20 minutes
4. If you haven’t done so already, add BME to SDS and then add prepared sample buffer
to samples
5. Boil samples for 10 minutes
6. Load samples and run PAGE gel for 1.5 hours at 120V
7. Set up transfer apparatus in a tray of blotting buffer (listed from bottom to top) Black
plastic- sponge- filter paper- gel- nitrocellulose membrane- filter paper- sponge-red
plastic. Remove air bubbles by rolling a glass rod over the top sponge. Remember
that protein goes towards the positive so make sure the membrane is between the gel
and the white plastic back
8. Put in 1X cold transfer buffer with ice pack and stir bar, matching black with black
9. Transfer at 120V for 1 hour in the cold room
10. Block in 20mls of 5% milk in TBS/Tween for 1 hour at RT or overnight in cold room
11. Incubate the primary antibody in 5% milk/TBS/Tween for 1 hour at RT or overnight
in cold room
12. Save primary antibody and wash the membrane 3X (15 minutes each) in TBST
13. Incubate with secondary antibody in 5% milk in TBST for one hour
14. Wash several times (at least 1 hour total) in TBST
15. Add ECC for 1 minute (use in a ratio of 1:40 aka 1ml of A and 25ul of B)
16. Add the plastic to the top of the membrane
17. Take film holder and film into the darkroom and add the film to the holder, exposing
for different time intervals
18. Develop film and compare to membranes in order to indicate the marker positions
19. To strip membrane incubate with 25ul stripping buffer for 30min at 50°C
20. Rinse with 1X TBST and start again from step 10