First thing to do--- check the pH of the water (I assume distilled,
deionized) that you added. Sometimes very pure water can have a rather low
pH (~5.5) if it sits and absorbs CO2 from the atmosphere over time. A little
bit of a buffer (Tris HCl, Hepes, or Na phosphate) might help. Salts (50-150
mM) may help the proteins form a better suspension or solution. Some protein
preps. contain a considerable amount of inert material that will not
dissolve--so the toxin component may actually be in suspension. Another
trick that sometimes works is to suspend the pertussis toxin by vigorous
vortexing and then quickly add and mix it in the cell culture medium that
you plan to use. The dilution plus the medium components (serum proteins,
etc.) will help the material to dissolve. You should pass the medium through
a 0.2 micron filter (preferably a low protein binding filter or pretreated
by pre-flushing with 1% bovine serum albumin) to remove undissolved
particles before exposing cells.
Best of luck,
Charles A. Miller, III, Ph.D.
Associate Professor of Environmental Health Sciences
Tulane University School of Public Health and Tropical Medicine
1430 Tulane Ave.
New Orleans, LA 70112
(504)585-6942 rellim at tulane.edu
Bionet.toxicology news group: http://www.bio.net/hypermail/toxicol/current
>> I want to use pertussis toxin to inactivate Gi family members in transfected
> COS cells to investigate some signaling pathways. I ordered the pertusis
> toxin from Calbiochem (cat. no. 516560), and it comes as a lyophilized
> solid to be reconstituted in water. Both the label on the vial AND the
> catalog description mention that I should obtain a "uniform suspension"
> before using the toxin on cells. I figured that should be easy enough.
>> As it turns out (to my surprise), the toxin forms a cloudy, particulate
> suspension when water is added to the lyophilized powder. Usually
> lyophilized proteins go into solution very quickly and easily, but not so
> here. I tried vortexing the hell out of it, but the clumps of toxin
>> I don't know if the toxin would stand up to sonication; and I don't want to
> add a detergent, since that will also get applied to the cells when I add
> the toxin to them.
>> Does anyone who routinely works with pertussis toxin have any suggestions on
> how to make a "uniform suspension"?
>hines at pharm.med.upenn.edu