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SDS Lysis

Whole-cell protein extraction of tissue culture cells can be obtained by using a simple buffer solution containing SDS (sodium dodecyl sulfate) and Tris base. The SDS acts as an anionic detergent that will cause cells to lyse and expell their nuclear and cytoplasmic extracts while the Tris base will keep the pH of the buffer stable. This method of protein isolation will not give a "pure" protein extract. If purity is important, more elaborate protocols must be used.

SDS Lysis buffer:

Basic protocol

1. Isolate tissue culture cells

2. Add 2mL of boiling lysis buffer per 15 mL centrifuge tube, swirling the solution over the cell pellet to enure rapid denaturation of cellular protiens.
3. The lysate sample will be viscous due to cellular DNA.
4. Transfer the lysate to a 50 mL centrifuge tube.
5. Briefly heat the lysate (heating block or microwave) with tube uncapped. This step should be monitered carefully, as there is a potential for sample loss due to spills.
6. One of these methods can be used to shear the cellular DNA:
7. Remove a 15 ÁL aliquot and add 1/5th volume (3ÁL) 6x SDS sample buffer, mix thoroughly and place in a boiling water bath for 2 minutes or heat block.
8. Store at -20C
I found myself nodding my noggin all the way through.

me 2 i have no idea what im doing pleas e help

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