[27]. The type of analysis will depend on the experimental
setup and research questions and should be determined by
each investigator.
8. A number of programs can be used to do these analyses
including R, JMP, SSPS, or Matlab for statistical analysis; the
R package SuperExactTest for the statistical analysis and visualization of multiset intersections [35]; DAVID [36, 37], Proteomaps [38], Panther [39], or GOrilla [40, 41] for gene
ontology enrichment analysis; and COMPLEAT for a subcellular localization enrichment analysis [27].
4 Notes
1. All fixation solutions must be made fresh, just prior to using
them. Do not store the solutions.
2. DTBP needs to be stored at 4
C but equilibrated to room
temperature prior to use (30 min). The stock solution of
50 mM will turn cloudy in approximately 5 min. Use the
stock before cloudiness occurs; otherwise, discard it. Reaction
pH is critical; basic conditions (pH 8–10) favor mechanism of
action of cross-linking by DTBP. It must be made fresh. Do not
store the solution.
3. RIPA buffer (without DTT and protease inhibitors) can be
stored at 4
C. However, DTT and protease inhibitors should
be added freshly before use.
4. 5Â Laemmli buffer can be stored for several months at À20
C,
but DTT should be added freshly before use.
5. Can be stored at room temperature for extended time but
protected from light. It might start to turn yellow over time,
but this color change does not affect its use.
6. Cells must adhere well to the membrane in order to isolate
them and/or cellular protrusions by LCM. However, whether
you use fibronectin, poly-lysine, collagen, or any other extracellular matrix protein coatings, you need to make sure that it
does not hinder the formation of the cellular protrusion of
interest.
7. MMI suggests that you seed the cells in the ring and later move
it to the UV-permeable microdissection chamber. However,
the most important issue when using an MMI live chamber is
to ensure that there is no leakage once the ring is attached to
the microdissection chamber. If the ring leaks and wets the
bottom of the membrane prior to putting inside the microdissection chamber, it will not properly attach to the adhesive area
of the chamber. As a result, the LCM-isolated cut will not stick
to it and will not be collected after removal of the ring. For this
Proteome of Cellular Protrusions Identified by LCM/MS
41
setup and research questions and should be determined by
each investigator.
8. A number of programs can be used to do these analyses
including R, JMP, SSPS, or Matlab for statistical analysis; the
R package SuperExactTest for the statistical analysis and visualization of multiset intersections [35]; DAVID [36, 37], Proteomaps [38], Panther [39], or GOrilla [40, 41] for gene
ontology enrichment analysis; and COMPLEAT for a subcellular localization enrichment analysis [27].
4 Notes
1. All fixation solutions must be made fresh, just prior to using
them. Do not store the solutions.
2. DTBP needs to be stored at 4
C but equilibrated to room
temperature prior to use (30 min). The stock solution of
50 mM will turn cloudy in approximately 5 min. Use the
stock before cloudiness occurs; otherwise, discard it. Reaction
pH is critical; basic conditions (pH 8–10) favor mechanism of
action of cross-linking by DTBP. It must be made fresh. Do not
store the solution.
3. RIPA buffer (without DTT and protease inhibitors) can be
stored at 4
C. However, DTT and protease inhibitors should
be added freshly before use.
4. 5Â Laemmli buffer can be stored for several months at À20
C,
but DTT should be added freshly before use.
5. Can be stored at room temperature for extended time but
protected from light. It might start to turn yellow over time,
but this color change does not affect its use.
6. Cells must adhere well to the membrane in order to isolate
them and/or cellular protrusions by LCM. However, whether
you use fibronectin, poly-lysine, collagen, or any other extracellular matrix protein coatings, you need to make sure that it
does not hinder the formation of the cellular protrusion of
interest.
7. MMI suggests that you seed the cells in the ring and later move
it to the UV-permeable microdissection chamber. However,
the most important issue when using an MMI live chamber is
to ensure that there is no leakage once the ring is attached to
the microdissection chamber. If the ring leaks and wets the
bottom of the membrane prior to putting inside the microdissection chamber, it will not properly attach to the adhesive area
of the chamber. As a result, the LCM-isolated cut will not stick
to it and will not be collected after removal of the ring. For this
Proteome of Cellular Protrusions Identified by LCM/MS
41