Biology Reference
In-Depth Information
4.3. Gel
Electrophoresis
and Incubation
7. We have found that the Gelatin Zymogram gels from Invitrogen
are the most sensitive in revealing gelatinolytic activity with an
incubation time of 6 days. While most commercial zymogram
systems should work equally well at the typical shorter (<24 h)
incubation periods, the longer incubation intervals (up to
6 days) we apply with our TBI samples require monitoring to
avoid possible bacterial contamination. With the Invitrogen
zymogram gels, we do not fi nd such contamination, making
them our fi rst choice for these experiments. If bacterial con-
tamination does occur during an extended incubation period,
sodium azide can be safely added to the incubation buffer to
stop bacterial growth (
3
).
8. Care must be taken that equal amounts of protein are loaded
since zymographic gel systems do not permit the standard load
control analysis used for Western blots. Gelatinolytic activity is
increased with protein load and may be used with a standard
curve approach; however, adding too much protein (generally
>50
g) can impair visualization of the lytic bands. Heavy
Coomassie blue stain intensifi es the adjacent proteins not
affected by MMP lysis and compromises on the clear visualization
of lytic bands (see Fig.
1
).
9. Each run should include corresponding sham-injured controls
so that experimental values may be expressed as percent of
control and allow standardization across multiple zymogram
runs within a study.
10. Measurement of gelatinolytic activity can be maximized by
incubation with the development buffer for extended length of
time for up to a week. The detection of gelatinolytic activity
in our tissue samples requires prolonged incubation with the
substrate for visualization of the lytic bands. The amount of
MMP standards should be adjusted accordingly to insure that
the gel lanes are not overly digested.
μ
4.4. Visualization
of Lysis
11. Zymogram gels should be destained carefully such that con-
trast between lytic signal and gel background is optimal for
densitometric measurement.
12. It is important to make sure that there is even illumination
across the gel when capturing and measuring density of the
lytic bands. Neutral fi elding should be applied to the gel image
capture step so that there is no artifact from your light source.
13. Other methods of measuring gelatinolytic activity, such as with
commercial kits employing fl uorescent peptide substrates, are
available to confi rm zymographic results. We have successfully
used a commercial kit for MMP-3 activity, and have tested
MMP-2 and MMP-9 kits with samples also measured by zymog-
raphy. To date, we have found that these kits may present their
4.5. Additional
Controls for MMP
Lysis
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