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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 14:40, 24 January 2007
, 24 January 2007→Hen Egg White Lysozyme
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===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 doesn't seem to allow it into solution 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 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 | 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 doesn't seem to allow it into solution 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 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. | |||
Lysis is apparent by a reduction in turbidity and a severe increase in viscosity from the liberated chromosomes. For protein preparations, I usually add Benzonase because it has activity toward double- and single-stranded DNA and RNA. If you just add DNase, be aware that most are purified from pancreas and are loaded with proteases. If you see a lot of damage to your protein during lysis, add proteiease inhibitiors and/or change nuclease. | |||
===Intracellular Lysozymes=== | ===Intracellular Lysozymes=== | ||
