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The following message was posted to: PharmPK
Dear All
I used SDS-PAGE to separate P-gp from cellular lysate , I used primary
antibody C219 , and used the chemiblot kit to develop it into color (
peroxidase based Kit ) , I'm getting nothing on the final PVDF developed
membrane.
I'm not sure if the transfer conditions( electroblotter ) was not
optimal.
How can I trouble shoot this.
In addition , is there any valid P-gp standard other than cell lines
that
overexpress P-gp?.
Thank you
Mohammad Shawaqfeh
Clinical & Administrative pharmacy
University of Iowa
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For SDS-PAGE you should use the prestained MW std (Biorad has some). If
they don't transfer you know something was wrong with the transfer.
Perodixase is pretty wimpy you want to switch to alkaline phosphatase
(perixdase dies within 20min and APHOS is stable).
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The following message was posted to: PharmPK
Dear Mohammad
You are probably aware but maybe ........... Did you methanol activate
your pvdf membrane?
Other then that there could be numerous reasons
Marker did not blot either? What works nice is to use some rainbow
marker, you can then to some extent assess whether HMW proteins also
blotted well.
What cell did you use? IF you can get your hands on LLCPK1-MDR1 cells or
MDCKII-MDR1 cells, they stably express the MDR1, you may have some sort
of positive control.
Hope it is of some help
regards
Hans
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The following message was posted to: PharmPK
Hi:
If you are doubtful about the transfer of proteins onto membrane from gel,
you can develop the membrane using Comassie brilliant blue at any stage,
even after your attempt with BCIP/NBT (I assume, you used this for
peroxidase based secondary antibody kit) colour development.
If you are using a molecular weight marker in your SDS-PAGE, that itself is
an indicator for the transfer of all your proteins. If you have any
clarifications about the general procedure, you please contact me.
Best wishes
Madhu
Ph. +44 (0)141 3313224 (Lab, Madhu)
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