Preparation of total cell lysates for immunoblotting

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The two most common methods of preparing samples for immunoblots are either to lyse the entire cell or tissue mass in sample buffer and load the total cell lysate on the gel or mix any protein sample 1:1 with double-strength sample buffer and load.

Preparing total cell lysates:
In this procedure, samples of cells or tissues are disrupted in an electrophoresis sample buffer. After the chromosomal DNA is sheared to lower viscosity, the samples are processed and run as usual for particular gel system. In the example given here, the samples are prepared for electrophoresis in a standard Tris/glycinE SDS-Polyacrylamide gel.
  1. Measure the volume of sample to be lysed. For tissue culture cells 109 cells = 1 ml = 1 gram. For tissues or organs, weighing the sample will be sufficient. 1 gram= 1 ml.
  2. Most samples should be boiled for 5 mins. Some antigen will come out of solution under these conditions; if no signal is seen, check different temperature.
  3. Soniate the sample to shear the DNA. Approx. 15-20 sec at full force is sufficient to shear the DNA.
  4. Spin the sample at 10,000 g for 10 mins. Recover the supernatant and discard any pellet. If necessary, determine the relative protein concentration. After the final spin, samples may be frozen at -200 c. They are stable at this step for months.
The samples are now ready for electrophoresis.
To visualize this protocol Click here

References: Antibodies- A laboratory manual; Editor: Ed Harlow and David Lane. Chapter-12 , Immunoblotting, Page no: 480-482.

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