Preparation of Gel Apparatus and Gel:

1. Gel apparatus is in the western blot cabinet in lab. Take the 15x25cm tray, transparent
tank, red plastic plugs and 16-well comb. But you can only image 10wells, so prepare your
gel accordingly!
2. Set up the system to be ready to pour the gel.
3. Gel preparation: 1.5% Agarose-0.1% SDS in 1X TAE buffer
 1.5 g Agarose
 125 ml 1X TAE buffer
 Mix agarose and buffer. Boil them in microwave.
 Add 1.25ml 10% SDS and mix well slowly, not foam.
 Pour into the gel tray.
4. Prepare Running Buffer: 0.1% SDS in TAE buffer
 7ml of 10% SDS
 700ml of 1X TAE buffer
 Mix and put into 4C fridge in lab.

Sample Preparation and Loading

5. Protein lysates are prepared differently according to purpose.
 Do not boil your samples after centrifugation.
 Mix 15ul sample with 5ul 4X sample buffer (2X TAE, 20% glycerol, 8% SDS,
bromophenol blue to preference, add fresh DTT to a final concentration of 1mM)
 Before loading, always spin shortly.
1. Put a little amount of running buffer onto gel to fill the wells.
2. Get the protein marker from -20C, Hi-Mark Prestained High Mol-Weight Std. Load 12ul
marker to the first well.
3. Load your samples, all 20ul.
4. Bring the apparatus to cold room. Put the rest of running buffer.
5. Run gel at 100V for 2h.

Transferring of Samples to Membrane

1. Prepare Whatman papers: Cut according to width of the gel
 Thick: 2 long pieces
 Thin: 2 short, as big as gel, pieces
2. Get two of small rectangular Pyrex. Wash before use.
3. Fill 1/3 of each Pyrex with Transfer Buffer: 150mM NaCl, 25mM Tris in 1X TBS at pH 7.5
4. Place them with a distance of gel tray apart from each other.
5. Get tissue paper (klenex). Divide into two and place in between Pyrexes so that they would
be as tall as Pyrex and as wide as gel tray.
6. Cut membrane (Protran BA 83) in the size of your gel.
7. After this point, you will prepare the transfer sandwich. For that, you will wet Whatman
papers, membrane and gel with transfer buffer.
  Wet one of the long thick Whatman papers and place the center part on tissue
papers and put each edge under the corresponding Pyrex.
 Wet and place one of thin Whatman onto sandwich.
 Wet and place your membrane onto sandwich.
 Wash your gel in deionized water and then in transfer buffer. Place it onto sandwich.
 Wet and place one of thin Whatman onto sandwich. Remove air bubbles by roller.
 Wet the long thick Whatman paper and place the center part on sandwich and put
each edge into the buffer in corresponding Pyrexes.
 Put gel tray on sandwich.
 Put a weight on sandwich, like 500ml filled bottles.
 Mix them all and put into 4C to make cold.
8. Let transfer happen overnight.