Biomedical Engineering Reference
In-Depth Information
10. Refer to the general safety precautions when dealing with
radioactivity.
11. We prepare the 2× import buffer with all the ingredients except
BSA, dissolve them in water and then adjust the pH with
KOH. BSA is added after adjustment of the pH. The purpose
of BSA in the buffer is stabilizing some precursor proteins.
Although other protocols suggest the usage of 3 % (w/v) BSA,
our laboratory found that this is generally not necessary. In
fact, the majority of precursors we tested does not need the
addition of BSA to be imported, or is even imported more
efficiently without BSA.
12. Valinomycin forms ionophores that dissipate the inner mem-
brane potential. Oligomycin is a potent inhibitor of the ATP
synthase. Antimycin A blocks transfer of electrons from cyto-
chrome
b
to cytochrome
c
.
13. Keep PK in single-use aliquots, and avoid multiple thawing-
freezing procedures as this abolishes the enzymatic activity.
14. When we perform a series of import experiments, we keep the
remnants of the preprotein aliquots at −20 °C and use them
mixed with a new aliquot the next day.
15. We keep mitochondria frozen at −80 °C in single-use aliquots
of 30 or 50 ʼL. It is recommended to thaw aliquots that are
needed quickly by hand warming and place them immediately
on ice. Use them as soon as possible after thawing, since the
quality of mitochondria strongly reduces if not kept at −80 °C.
16. To test for spheroplast formation, add 50 ʼL cells to 2 mL
water or sorbitol buffer. The suspension in water should clear
up as an indication that the cells are being lysed in this hypo-
tonic solution. This effect can be also detected by measuring
the OD
600
and comparing the optical densities from both
suspensions.
17. The water used for RNA synthesis should be sterile and of pur-
est quality. In addition, the yield of RNA can be increased by
linearization of the DNA template prior to transcription. This
is particularly advisable when T3 or T7 polymerases are used
instead of the SP6 polymerase.
18. RNA produced in this way can be stable for more than 1 year.
19. Lower molecular weight translation products are often
observed in addition to the desired full-length product. This is
due to the initiation of translation at internal start codons
which can be suppressed by increasing the concentration of
Mg
2+
and K
+
ions during the translation reaction. However, in
most cases an internal initiation is irrelevant since N-terminally
truncated proteins lack their mitochondrial targeting signal
and will not even bind to receptors of the outer mitochondrial
