Biomedical Engineering Reference
In-Depth Information
the gold. Shake at RT for 15 min,thenadd0.2ml Soln. B to each
Determination of
optimal dilution
tube.Thecoloroftheredgoldsolswitchestoblue,iftheamount
of protein is not sufficient. Read at 650 nm and plot O.D. against
amount of protein. The optimal amount is that dilution which gives
just no color change.
Dilute the protein solution to a concentration corresponding
to the 1.5-fold of the optimal dilution. Pour 1 vol.ofthediluted
protein into a centrifuge tube and quickly add 5 vol. of dialyzed
gold colloid. Vortex and incubate at RT for 15 min.Add0.1vol.
Soln. C per 10 ml protein gold mixture and vortex again.
Spin with 11 000
g at RT for 30 min and resuspend the pellet
into a volume of Soln. D corresponding to the original volume of
the gold sol. Spin again and carefully resuspend the pellet in 1/10
volume of Soln. D.
The red solution is stable at 4
◦
C for 2-3 weeks. If sediment
occurs, spin with 500
×
g for 5 min and discard the pellet.
For staining of Western blots use a 1:250 dilution in PBS.
×
References
Geoghegan WD, Ackerman GA (1977) J Histochem Cytochem 25:1187
Goodman SL, Hodges GM, Livingston DC (1980) Scanning electron mi-
croscopy 1980 II:133
4.2 Immunization of Laboratory Animals
µ
Use 50 - 200
g of antigen (if conjugates are used: of hapten) depen-
dent on species and antigenicity per injection for immunization.
If KLH conjugates are used, there is no need for adjuvants. In all
other cases, mix the antigen solution with a commercially avail-
able adjuvant (the well-known Freund's complete and incomplete
adjurant should not be of first choice since it often induces severe
necrosis). For the ratio of antigen solution to adjuvant, read the
instructions for use given by the supplier. If the antigen is pre-
pared by electrophoresis, mince the cutted gel band thoroughly in
a small volume of PBS and immunize by intramuscular injection
Immunization
using PAGE samples
of the suspension into the hind legs nearby the lymph nodes or
intradermal injection into the back. To treat animals with care, do
not apply more than 150
µ
l of gel suspension per kilogram body
weight. Never use suspensions or emulsions for intravenous in-
jection! Especially when solutions are injected intravenously, filter
these solutions through a 0.22-
µ
m sterile filter.
As conjugates, complexes from biotinylated hapten and strep-
tavidin may be used. In that case perform the detection with avidin-
biotinylated hapten because avidin and streptavidin do not cross-
react.
Mix 300
µ
l of antigen or hapten-carrier conjugate in PBS or TBS
(without sodium azide or Thimerosal and without SDS) with 1 ml
