Reagents
Cold methanol (MeOH)
Transfer buffer (TB)
Procedure Wear gloves to avoid contaminating membranes
Rinse gels in transfer buffer
Wet membrane with MeOH
Then prewet membrane in transfer buffer
Saturate filter paper and sponges in transfer buffer
Fill Glass Dish with ice cold TB
Lay Clear/Black Press open, black side down in dish
Stack the transfer in the following order
1.Blackside of press (negative side)
2.Sponge
3.Filter paper
4.Gel
5.Membrane
§Make sure there are no bubbles under membrane
6. Filter paper
7. Sponge
8. Clearside of press (positive side)
Clamp press together under TB (=”Press-stack”)
Place the mini Trans-blot (red and black) electrode into Bio-Rad Tank
Fill Bio-Rad Tank with TB
Add stir bar to bottom of tank
Add ice block to side
Add Green lid
Place press-stack in Red and Black electrode (clear/red and black/black)
Fill tank with TB
Place tank on stir plate and turn on to 6-7 at 4C (i.e. cold room)
Plug into power supply, turn on, 100 V (+ 3V ok) x 60 min
After transfer:
Open clamp and remove membrane carefully with forceps
NO HANDS ON MEMBRANE
Block nonspecific binding sites by placing membrane in tray with 20 ml 5%milk/PBS
Block for 1 hour at RT, or overnight at 4°C
Primary Antibody incubation
Make appropriate dilution of antibody in 5% milk/PBS
Incubate I hour at 37°C or over night at -4°C
Wash 3 x PBS-05% Tween for 10 min
Secondary antibody Incubation
HRP-conjugated secondary antibody (generally anti-mouse or anti-rabbit, depending on your primary antibody species)
Make appropriate dilution of antibody in 5% milk/PBS
Incubate 1 hour @ 37°C
Wash 3x PBS-05% Tween for 10 min.
Develop with enhanced chemiluminescence (ECL) technique (Amersham)
Add ECL reagents to 1.5ml microcentrifuge tube:
1000µl Reagent A
25µl Reagent B
Pipette ECL reagents onto membrane and incubate for 5 min in dark
Do not allow membrane to dry
Remove excess liquid by tapping edge of membrane on paper towel
Put membrane protein side face down on plastic wrap and cover – NO BUBBLES
Put membrane inside film cartridge (protein side up)
In Dark Room, place film on top of membrane and develop after x min.
