比较蛋白质组学

Materials
Ready Gel Precast Gels
Bio-Rad Mini-Protean-3 cell apparatus
Prestained protein markers

Reagents
6x Laemmli reducing sample buffer
Distilled H20 (dH20)
Running buffer
Cold Transfer buffer

Before you start:
Turn on heat block to 95-100°C
       Make sure ice block cooling unit is prepared for transfer

Procedure
1. Prepare Ready Gel Cassette:
Remove the Ready Gel from storage pouch
Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer
Cut along the dotted line at the bottom of the Ready Gel cassette with a razor blade
Pull the clear tape at the bottom of the Ready Gel Cassette to expose the bottom edge of the gel
2. Assemble Electrophoresis Module (see Mini-Protean 3 cell assembly guide)
3. Sample preparation:
a.Total volume to be loaded: 30ul
x ml of sample (use 30ug total protein/well)
5 ml  6x Leammlis sample buffer
y ml dH20 (for a total volume of 30ul)
b. Heat samples @ 95°C for 5 min. briefly spin
c. Load protein marker to end well
d. Load 30µl sample with loading tips  
Note: carefully load lanes so samples don’t spill into adjacent lanes
4. Gel Electrophoresis
Mini Tank Assembly
Put Green lid on (red/red and black/black)
-  Power Supply
oTurn ON
o Constant Volt
- Run @ 120V ~10min (get samples through stacking gel)
- Run @ 100V until marker is near bottom of gel (~1.5 hrs)

Transfer
Additional Materials
Glass Pyrex Dish
Clear/Black sandwich Press
Sponges
Whatmann filter paper
Forceps to handle membrane
Nylon Membrane
                          NO HANDS ON MEMBRANE

Cut filter paper and membranes to size
Ice block (located in–20°C freezer)
Small stirring bar
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