Modified protocol for Western blotting

A. Preparation of cell lysates

1.    Harvest cells (70-85% confluent) by trypsinization and spin.

2.    Lyse the pellet with 100 µl lysis buffer 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.

1.    Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 20 min at 4°C.

2.    Transfer the supernatant to a new tube and discard the pellet.

3.    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.

1.    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.

1.    Boil for 5 min.

2.    Cool at room temperature (RT) for 5 min.

3.    Briefly spin to bring down the sample and loading buffer mixture prior to loading gel. 

refer to: http://www.e-biotek.com/protein/244-western-blot-protocol.html