Lysis experiments of cultured animal cells, yeast and bacteria for immunoblotting

Summary

Source: Laboratory Guide to Proteins and Proteomics

Operation method

basic program

Principle

The descaler lysis cell method is typically used for cultured animal cells. Typical ionic descaler SDS (e.g. 2% SDS) lyses the cells sufficiently. Cultured animal cells and bacteria, for example, can be lysed in this way. If the antigenic determinants recognized by the antibodies used in the experiment depend on the natural spatial conformation and are sensitive to reducing environments, dithiothreitol should be removed from the lysis buffer and a non-reducing/urea-free gel is recommended.

Materials and Instruments

Cell cultures, bacteria or yeast cells in suspension or monolayers
Dithiothreitol (DTT) 1 x Laemmli sample buffer
Centrifuge Water bath

Move

① Add ^1X107 ~ ^5X107 cells to 1ml of Laemmli sample buffer containing 100mmol/L DDT.

② Due to the release of DNA, the cell lysate becomes sticky in Laemmli buffer, there are 2 ways to overcome the stickiness problem:

a. Centrifuge the lysate at 100,000 g for 20 min to remove the DNA; and

b. Ultrasonication to break the lysed cells, 15~30s per ultrasonication, 4 times in total, and place the sample on ice for 15s between each time.

③Heat the samples at 95℃ water bath for 5min.

④ Centrifuge at 20000g for 5min.

⑤ Transfer the supernatant to a new tube.

⑥ The sample (supernatant) can now be used for electrophoresis and immunoblotting. In immunoblotting studies, when preparing extracts of cultured cells, protein samples must meet the following conditions:

a. be dissolved in a solution suitable for the gel electrophoresis system (e.g., the solution should have a pH of about 7.0 and a salt concentration of about 200 mmol/L);

b. The protein concentration should not exceed the carrying capacity of the particular gel system. For conventional gels, the sample volume per lane for small gels should not be >150 μg of total protein.

Caveat

1×Laemmli sample buffer:2% SDS10% glycerol60mmol/L Tris-Cl (pH 3.8)0.01% Bromophenol BlueIt is usually easier to make Laemmli Sample Buffer into a 2X or 5X reservoir. Add DTT to a final concentration of 100 mmol/L before use.

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