WESTERN PROTOCOL

A. Preparation of cell lysates

1. Collect cells (confluent T-25) by trypsinization and spin.
2. Lyse the pellet with 100 µl lysis buffer on ice for 10 min.
   For 500,000 cells, lyse with 20 µl.
3. Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 10 min at 4°C.
4. Transfer the supernatant to a new tube and discard the pellet.
5. Determine the protein concentration (Bradford assay, A280, or BCA)
   (We use the Bradford assay from Bio-Rad.)
6. Take x µl (= y µg protein) and mix with x µl of 2x sample buffer.
7. Boil for 5 min.
8. Cool at RT for 5 min.
9. Flash spin to bring down condensation prior to loading gel.

B. Polyacrylamide gel (14.5 cm x 16.5 cm)

1. Agarose plug:
   1% agarose dissolved in 1x Resolving gel buffer.
   (I make 50 ml, keep melting it as I need it, and re-adding water to maintain agarose conc.)
2. Resolving gel: 24 ml of a 9% gel
   5.4 ml 40% acrylamide/bisacrylamide (29:1 mix)
   3 ml 8x Resolving gel buffer
   15.6 ml water
   12 µl TEMED
   60 µl 20% ammonium persulfate
3. Stacking gel: 8 ml
   1 ml 40% acrylamide/bisacrylamide (29:1 mix)
   2 ml 4x Stacking gel buffer
   5 ml water
   8 µl TEMED
   21.6 µl 20% ammonium persulfate

C. Preparation of gel

1. Assemble the glass plates and spacers (1.5 mm thick).
2. Pour an agarose plug (1-2 mm).
3. Pour the running gel to about 1 cm below the wells of the comb (~20 ml).
4. Seal with 1 ml water-saturated 1-butanol.
   (Can stop here and leave gel as is overnight if you want.)
5. When gel has set, pour off the butanol and rinse with deionized water.
6. Pour the stacking gel (~5 ml) and insert the comb immediately.
7. When the stacking gel has set, place in gel rig and immerse in buffer.
8. Prior to running the gel, flush the wells out thoroughly with running buffer.