Modified Protocol for Western Blotting (Page 1,  2,  3)--Western Blotting Center

A. Preparation of cell lysates

1. Harvest cells (70-85% confluent) by trypsinization and spin.
2. Lyse the pellet with 100 µl lysis buffer per million cells on ice for 10 min.

Note: 1) Just scrub the cells after washing with PBS and adding the lysis buffer, if the cells being plated at dishes or plates.

2) Sonication is optional after lysing the sample, but just don’t sonicate the sample too long. We suggest it should be sonicated for no more than 10 seconds.

3) The amount of lysis buffer is flexible. It depends on the number of cells you have.

4) You need to homogenize it first if you do Western blotting with tissue.

3. Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 20 min at 4°C.
4. Transfer the supernatant to a new tube and discard the pellet.
5. Measure the protein concentration with protein assay whichever method set up by your lab. (e.g. Bradford assay, or BCA; or Lowry Assay)

Note: Don’t dilute the protein too much. You can concentrate the protein lysate with vacuum under 4ºC in case you got a very low protein concentration.

6. Take the same amount protein lysate from each sample separately, and mix with sample loading buffer. The final concentration of loading buffer must be 1X.

e.g. The ratio of sample (ul)/ 2X loading buffer (ul) should be 1:1. The ratio of sample (ul)/ 6X loading buffer (ul) should be 5:1.

7. Boil for 5 min.
8. Cool at room temperature (RT) for 5 min.
9. Briefly spin to bring down the sample and loading buffer mixture 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 you 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.