18
Various methods can be used to measure the efficiency of the disruption. For example, a
visual count can be made using a microscope.
For greater accuracy, a protein assay could be used. This procedure is widely recognized
as a good method for measuring cell disruption by taking into account the amount of
protein released after disruption. The disrupted cells are then tested and checked against
this number for percentage breakage.
There are several types of protein assays. One commonly used is the Folin Reaction
(Lowry Assay) method, as it is comparatively simple and provides consistent results.
This colorimetric method has a sensitivity to protein of around 8 µg / mL in the assay
solution.
The assay turns blue in the presence of proteins due to the reaction of copper ions in the
alkaline solution with protein and the reduction of phosphomolybdate- phosphotungstic
acid in the Folin reagent by aromatic amino acids in the treated protein.
Fractional protein release, Rp, is calculated using the following equation and multiplying
the result by 100:
Rp = Cf – Cb
Ct – Cb
Cf = Free protein
Ct = total protein
Cb = Background protein
This gives the actual disruption percentage, taking into account the background levels of
protein before disruption.
Since the greatest concentration of energy is beneath the probe, it is imperative that the
sample be kept as close to the tip as possible, liquids are easily processed because the free
moving cells circulate repeatedly below the probe. Solid materials however have a
tendency to be repelled by the ultrasonic, and should be processed in a vessel large
enough to accommodate the probe, yet small enough to restrict sample movement. For
small samples, conical shaped test tubes are recommended.
Allowing the probe to contact the vessel will decrease the power output, and cause
minute grey glass particles to migrate into the sample. Although these glass particles will
not adversely affect the chemical composition of the sample, they will form a thin grey
layer on centrifuging. If the probe has to come in contact with a solid sample, use a
standard 20mm (3/4”) diameter stainless steel centrifuge tube cut to 70mm (3”) length.
Do not use a glass tube. Microtips must never allowed to come in contact with anything
but the liquid, because the stress resulting at the point of contact with a hard surface will
cause the microtip to fracture. Although larger probes will not fracture if they come in
contact with a glass vessel, they may cause the vessel to fracture.
Before each application, place the tip in water or alcohol and energize the power supply
for a few seconds to remove any residual substances. If concerned about contamination
from previous use, clean the probe with a 20% Virkon solution and rinse with distilled
water. For critical application, probes may be autoclaved.
(16 pages)
(12 pages)
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