Biomedical Engineering Reference
In-Depth Information
stock solution should be prepared in water and stored at 4°C.
APS decomposes slowly. Fresh solutions should be prepared
weekly.
5. TEMED may decline in quality after opening (gels will take
longer to polymerize).
6. Transfer buffer can be used for two transfers within 1 week.
7. Tween-20 is a viscous solution and causes problems during
pipetting, partially avoided by cutting the pipette tip.
8. Once diluted, the primary antibody can be stored at −20°C
for up to 3 weeks. In addition, the primary antibody can be
used in three different blots with the only adjustment required
for increasing length of exposure to film at the ECL step.
9. To avoid changes in pH due to the high alkalinity of the per-
oxynitrite stock solution, an appropriate amount of 1.5 N
HCl, approximately the same amount of the peroxynitrite
stock solution, normally made in 1.5 N NaOH, was also
added to the wall of the tubes prior to peroxynitrite. Note
that this procedure has to be fast since peroxynitrite rapidly
decomposes at neutral pH.
10. Note that the numbers of passages necessary to lyse the cells
may differ significantly among different cell types. The status
of the cells can be conveniently monitored after addition of
trypan blue under a microscope.
11. The fractionation procedure of U937 cells normally yields
about 1.7 mg/ml proteins for the cytosolic fraction and
1.2 mg/ml proteins for the mitochondrial fraction.
12. Apply 5-20 mg proteins to each well of a 0.75-1.0 mm thick
gel. For thicker gels (i.e. 1.5 mm), apply up to 25-40 mg in
each well.
13. A syringe is normally used for 0.75-1.0 mm thick gels. Use a
micropipette for 1.5 mm thick gels.
14. Interrupt the experiments if colored molecular weight mark-
ers are not clearly visible on the membrane. It indicates that
something did not work during transfer.
15. Apply a luminescent sticker on the edge of the acetate
sheet to provide an alignment mark for the film and the
membrane.
Acknowledgments
This work was supported by grant from AIRC and PRIN (OC).
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