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Sauer:Lysing E. coli with Lysozymes: Difference between revisions
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Sauer:Lysing E. coli with Lysozymes (view source)
Revision as of 14:51, 24 January 2007
, 24 January 2007→Getting The Most Out Of Your Bugs
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Submitted by [[User:Smoore|Sean Moore]] | Submitted by [[User:Smoore|Sean Moore]] | ||
==Getting The Most Out Of Your Bugs== | ==Getting The Most Out Of Your Bugs== | ||
Native lysis is a staple protocol in practically every biochemistry lab, yet there is significant variability in the procols I have read. The intention is to liberate the guts of E. coli without disturbing the native conformations of the biomolecules. Using a French pressure cell you can mechanically disrupt the cell using a sharp drop in pressure, it is a great way to lyse cells. Unfortunately, it is impractical for multiple samples or small-volume samples that don't fit in the pressure cell. Besides, the machine is scary, lysis can be variable, and cleanup is a hassle. Chickens and bacteriophage have evolved great a way of opening E. coli using enzymes. Most commercial lysozymes are free from proteolytic activity and can be added in large amounts. Be careful, if your protein is ~14 kDa you may inadvertantly purify the added lysozyme instead of your target protein. Don't laugh, it's happened more than once and I have met people who solved the crystal stucture of lysozyme by mistake. | |||
===Hen Egg White Lysozyme=== | ===Hen Egg White Lysozyme=== | ||
I prepare the lysozyme fresh each time from lyophilized powder. Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened. Pure B-Per | I prepare the lysozyme fresh each time from lyophilized powder. Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened. Pure B-Per and most lysis buffers don't seem to allow it into solution well and it remains mostly as an inactive precipitate. So I make a ~10 mg/mL stock in some buffer off my shelf (I usually use 50 mM bis-Tris, pH 6.5). Then I dilute from this to 0.1 to 0.01 mg/mL in the B-Per lysis solution. To save time, I use a 200 μL pipette tip and lift out dried lysozyme horizontally from the bottle, I then place a microfuge tube over the tip and shake off the powder. I measured the before and after weight of several tubes and found that I can make pretty close to a 10 mg/mL solution by just adding 400 μL of buffer to the tube. So, I no longer weigh out lysozyme each time, I just assume that my tip carries about 4 mg of protein. | ||
In most cases, simply adding lysozyme to your resuspended cells and waiting is sufficient to invoke lysis. Some protocols call for placing the cells on ice during lysis in an effort to curb proteolytic activity. This never made much sense to me, you also slow down the activity of the lysozyme. I have never seen a comparison of the activities of proteases and lysozyme at 0 and 25 degrees, but I generally lyse at room temperature. | In most cases, simply adding lysozyme to your resuspended cells and waiting is sufficient to invoke lysis. Some protocols call for placing the cells on ice during lysis in an effort to curb proteolytic activity. This never made much sense to me, you also slow down the activity of the lysozyme. I have never seen a comparison of the activities of proteases and lysozyme at 0 and 25 degrees, but I generally lyse at room temperature. | ||
