endogenous phosphatase

C Jamison jamisoncsNO at SPAMaccessam.com
Sun Jul 25 06:20:07 EST 1999


Paula:

It would help me if you described your method a little more.  I assumed that
you were simply looking for positive or negative plaques.  I am also
assuming that these are lambda phage and you have infected E. coli for your
plaque lifts.  Are you getting light all across the entire membrane?  or are
you only getting light where there are plaques?  Is the light amount
proportional to the size of the plaques?  If there is light all across the
membrane, then you need to block the membrane better, or change membranes as
I suggested in the last post, or your x-ray film exposure is too long.  If
you are getting light in all the plaques, then either all your plaques are
positive, or your X-ray film exposure is too long and the background is
catching up with the positive results.  You may need to do very short
exposures with the X-ray film, or perhaps dilute the detection substrate.
The x-ray film detection may only require a second or 2 of exposure, and
that is why I used the colorimetric substrates; the chemiluminescent methods
are more difficult to control, in part because it is difficult to place the
x-ray film on the membranes (separated of course by a piece of plastic wrap)
for that short a period of time and repeat that procedure in a consistent
manner.  (As an aside comment, I and two other independent researchers in
our Dept. tried chemiluminescent procedures and were never happy with the
inconsistent results.)  A final set of questions: how does the colorimetric
method compare to the chemiluminescent results?  Does the coloimetric method
work and the chemiluminescent not work or vice versa?

>...including phosphate in the medium...
1)  I have never worried about adding phosphate to the medium in these kinds
of experiments, I simply grew the cells on standard media (LB, LB+glucose,
LB+maltose, etc.).  E. coli has a *relatively* low level of phosphatase,
meaning that:  so as long as your insert expresses more than E.coli you wil
be okay.  If you are looking to eliminate the E. coli phosphatase
background, then you may have a difficult road ahead.

>...washing in PBS.
2)  In general, I used BSA with PBS to block membranes and used BSA (0.1%)
with PBS for washes and adding antibodies.

Scott Jamison

Paula Murphy wrote in message ...
>> Also, try a colorimetric method for detection; it is often faster and
>>easier to control, with less background
>>than the chemiluminescent methods.
>> Scott Jamison, Ph.D.
>
>Well you see, sensitivity is actually the point of this small experiment.
>My supervisor wants me to use chemi. and I am struggling to find a way to
>knock out the endogenous AP. I am concurrently using colorimetric methods
>but only for comparison sake. No one seems to have commented on my
>question about including phosphate in the medium and washing in PBS. Is
>this because it is not likely to work?
>
>Thanks,
>Paula Murphy





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