I would like to measure lysozyme activity in turbot larvae. Has anyone experience in what is the minimum wet weight that can be used to assess the activity with the Micrococcus turbidity assay in 96-well plates? I have tried to use this assay but don't see any change in absorbance so far even though the positive control works fine.
Dear Dr. Miest
First of all you must homogenize the larvae to measure lysozyme in extract.
Pooled larvae should be homogenized in 4 parts of phosphate buffer (50mM; PH: 6.2). Then you should centrifuge it at 30000 g for 30 min (4°C). After that, separate the supernatant. Then, supernatant should be subjected to acidic precipitation with 2.5 M acetic acid (PH: 4.5) and centrifuge it at (10000 g; 15 min; 4°C). Supernatant should be neutralized with NaOH 1 M then subjected to fractional precipitation with (NH4)2SO4 (85%) at 4°C. finally, you should redissolve the 85% precipitate in 50mM Tris/HCL buffer (PH: 7.4) to bringing a clear solution. This solution must be desalted and should equilibrated to the assay buffer (0.02 M sodium phosphate buffer (SPB), pH 5.8) using PD-10 column.
For turbidimetric assay: using lyophilized Micrococcus lysodeikticus (Sigma, St Louis, MO, USA) was used to determine lysozyme activity in supernatant. 135 µl of M. lysodeikticus at a concentration of 0.2 mg ml-1 (w/v) in 0.02 M sodium phosphate buffer (SPB), pH 5.8 was added to 15 µl of serum sample. As a negative control, SPB was replaced instead of plasma. Results were expressed in units of lysozyme ml−1 supernatant.
A unit of lysozyme activity was defined as the amount of supernatant causing a reduction of 0.001 per min at 450 nm at 22 ºC.
Dear Dr. Torfi,
thank you for your answer. It helps a lot. What is your recommendation how many larvae should be pooled?
Dear Dr. Miest
Hi
Your welcome. When we use tissue for enzyme assessment, we usually use around 0.5 to 1 gr of wet tissue. If you use 1 Day post hatch larvae, please homogenize it with this procedure because it has soft tissue; using a homogenizer at 10000 rpm for 3 pulses of 30 s each with an interval of 30 s between each stroke (besides ice).
Best wishes
Mansour
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