Total proteins are to be extracted from Adenovirus-infected A549 cells. Pellet sizes range from roughly 1,000,000 cells to 4,000,000 cells.
What would be the ideal amount of lysis buffer (Tris-HCl, SDS, Glycerol, Bromophenol blue) to add to each sample in order to make sure that total proteins have indeed been extracted? Adding a large amount of lysis buffer would ensure full protein extraction but would defeat the purpose, as the now ultra-diluted proteins would be rendered unusable for SDS-PAGE. But what about adding too little lysis buffer? How can one effectively know if proteins were extracted below 100% efficiency?
You can lyse the cells in a larger volume to ensure the best efficiency in lysis and once it is done you can TCA preciptate your total protein and resuspend it in a smaller volume for loading in a gel. Make sure that the acetone is completely boiled off and resuspend your TCA preciptated pellet in the alkaline SDS loading buffer to ensure your pellet is compleltey dissolved. The process takes about 30 to 45 minutes of extra time.
As for the ideal volume of lysis buffer, there isnt an optimal ratio that I know of but 100uL/8 million cells sounds reasonable. But if you are planning to TCA preciptate, you can increase the buffer volume as much as you want. And as for buffer composition, if you are planning to do just SDS-PAGE for separation and Western Blots, consider using RIPA buffer.
In addition to Praveesh Valissery , SDS will dissolve the nuclei (in contrast to non ionic detergents like Tween 20 or TX100) and your extract will become quite gooey and difficult to pipet. You may shear the DNA by vigorous pipetting or passing the extract through a narrow needle, e.g. by using a syringe, e.g the kind that is used for injecting insulin.
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