(Criterion gel system from Bio-Rad)
General Preparation

Freeze ice-block before use

Make 1 L running buffer o 1X Tris/Glycine/SDS, from 10x (Bio-Rad #161-0732)

Make and chill transfer buffer at 4°C (improves heat dissipation) prior to running gel: o 1X Tris/Glycine/20% methanol buffer (2 L)

6.06 g Tris

28.8 g Glycine

400 ml methanol

Add dH
2
O up to 2 L
Sample Preparation
1.
In hood, mix 950
 l of Laemmli sample buffer (Bio-Rad #161-0737) with 50
 l of

mercaptoethanol, and pipet up and down a lot to mix.
2.
To prepare the sample, add 1 part sample and 1 part Laemmli/

-mercaptoethanol mix.
For instance: a.
For each sample, prepare 20
 g of total protein in a final volume of 22.5

L. b.
Add 22.5
 l Laemmli/

-mercaptoethanol mix. Final volume= 45
 l
Always keep the stock protein samples and the loading protein samples on ice until ready to boil. Spin the protein samples for 3 minutes at 4°C to precipitate any particulate. Pipette the proteins very slowly.
Running the gel
Set-up
3.
Remove gel cassette from storage container and remove tape from bottom of cassette
(gel is 4-20%; Bio-Rad #345-0032)
4.
Insert each gel into one of the slots in the tank, with the upper buffer chamber facing the center of the apparatus.
5.
Fill the upper buffer chamber in each gel with 60 mL of 1X running buffer.
6.
Gently remove the comb, and use a dropper to rinse and remove bubbles from wells.
Loading
7.
Boil prepared samples for 5 minutes; then keep on ice until ready to load!!
8.
Load 45

L of sample mix into wells using gel-loading tips. Load 15

L of
Kaleidoscope protein marker (Bio-Rad #161-0324) in first well.
9.
Fill the tank to the line molded into the sides of the tank (~800 ml)
Updated 03/05/14 by CM 1

Start-up
10.
Put lid on tank, insert leads with proper polarity, and run gel at 150 V (low-range) for approximately 60 minutes. Can increase the voltage up to 200V, depending on smileeffect; and decrease running time, depending on how far the dye has traveled.
Gel Removal
11.
Turn off power supply, remove lid, lift out cassette, and discard the running buffer.
12.
Break weld-joint on gel cassette by using the cassette-opening tool built in to the lid of the tank, gel comb, or metal spatula. Pull the 2 cassette halves apart.
13.
Cut the top and bottom of the gel to remove the wells and the bottom overthickness.
14.
Invert the gel and plate under transfer solution and agitate gently until gel floats off the plate. Or, gently lift the gel and place into container filled with transfer buffer.
Incubate gel in transfer buffer for 15 minutes.
Transfer:
Preparation for blotting
15.
Cut nitrocellulose membrane (Bio-Rad #162-0148) or PVDF membrane (Bio-Rad
#162-0175) to filter paper (Bio-Rad #1704085) dimensions.
16.
Slowly and gently wet the membrane in ddH
2
O (nitrocellulose) or 100% methanol
(PVDF). The PVDF membrane should have a gray, translucent appearance.
17.
Set up gel/membrane sandwich: a.
Pour chilled transfer buffer into each compartment of gel/blot assembly tray b.
Place the cassette in the back/large compartment of the tray: open the cassette so that the red side with the handle is vertical (anode) and the black side
(cathode) is lying horizontal and submerged in the transfer buffer c.
Place a fiber pad on top of the black side of the cassette, submerged in buffer.
Push on the fiber pad with a gloved finger to thoroughly soak the pad d.
Place a piece of filter paper on top of the fiber pad (it will wet immediately) e.
Gently place the pre-equilibrated gel UPSIDE-DOWN on top of the filter paper. Use roller to remove any air bubbles that may be trapped underneath the gel. f.
Place the membrane in the front/small compartment of the tray and let it equilibrate in transfer buffer. Use clean gloves and place it on top of the gel, taking care not to trap any air bubbles. The membrane should not be moved or adjusted once it touches the gel. Use the roller to roll out bubbles g.
Place a piece of soaked filter paper on top of the membrane, and remove air bubbles h.
Wet a second fiber pad in the front compartment of the tray, again using fingertips to completely saturate the pad with transfer buffer. Then place the wet fiber pad on top of the second filter paper. i.
Lower the clamp-slide of the cassette, and lock in the closed position.
Updated 03/05/14 by CM 2

Set-up of Gel Sandwich
Startup of Transfer
18.
Fill the blotter tank with transfer buffer to ~50% of fill volume
19.
Place a magnetic stir bar in the tank
20.
Place the ice block in the ice block pocket in the back of the cell; flip down lever to hold it down
21.
Move the locked cassette into the groove in the blotter tank, aligning the red side of the card with the red electrode. Make sure the magnetic stirrer is free to move.
22.
After both cassettes are in place, add the remaining transfer buffer to the fill level marked on the tank
23.
Put on the lid, plug the cables into the power supply, and run the blot for ~60 minutes at 100 V. perform run on ice AND in the cold room, on top of magnetic stir plate.
24.
When run is completed, disassemble the blotting sandwich and remove the membrane for total protein detection.
Total Protein Detection
25.
Wash the membrane 3 times (5 minutes each) with ddH
2
O.
26.
Stain the membrane using approximately 50mL of Ponceau S solution (Sigma P-7170,
1L Ponceau S Solution) during 5-10 min at RT on a rocker plateform. The Ponceau s solution can be reused 3-4 times.
27.
Rince the membrane with ddH
2
O in order to clearly see the stained bands.
28.
Take a picture using the Biorad Chemidoc (settings: blot/colorimetric).
Updated 03/05/14 by CM 3

29.
Unstain the membrane: wash 3 times (5 minutes each) with TBS/T solution (wash buffer) (1X TBS/0.1% Tween-20).
Wash Buffer (1X TBS/0.1% Tween-20) [TBS/T] (might be stored for a day in the cold room)
For 1000 mL:

100 mL 10X TBS

1 mL Tween-20 (add while stirring)

Add ddH
2
O up to 1000 mL
30.
Rinse all parts of apparatus with dH
2
O.
31.
Can store membranes in Ziploc bag at 4°C, or proceed to immunblotting protocol.
Kaleidoscope protein marker (Bio-rad 3 161-0324)
Updated 03/05/14 by CM 4