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The following message was posted to: PharmPK
Dear All
The western blot for the P-gp in mice liver give out a two
distinct bands.
I'm not sure if these are two forms of P-gp. Any clue ?.
I'm going to apply the same to rats , Is it possible to use the
same
Antibodies for the mice settings?
Thank You
Mohammad Shawaqfeh
University of Iowa
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The following message was posted to: PharmPK
Mohammad,
Mature P-gp is 170 KDa protein which is incorporated
in the membrane after cellular trafficking of a
precurssor P-gp protein of 120 KDa size. During golgi
to membrane traficking , post-translational
modification occurs that involves N-terminal
glycosylation resulting an increase in the molecular
weight.
Hence, often 2 bands are observed ~170 KDa and ~120
KDa while doing westersn. Careful sample prep i.e.,
adding higher conc. of surfactant and heating the
samples atleast for 5 mins reduces the immunoreactive
band corresponding to Pre-P-gp.
You did not mention about the antibody used. However,
an antibody raised against an antigen corresponding to
the N-terminal of P-gp is preferable since it
predominantly recognises the mature P-gp i.e., the 170
kDa band.
Furthermore, you could eliminate the presence are
Pre-P-gp by extracting plasma membranes by
density-gradient based centrifugation instead of using
the whole cell protein extracts for western blots.
Hope this helps !
Regards,
Santosh
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Dear Mohammad,
Regarding your first question, I guess the following reference will
help.
Tang F, Horie K, Borchardt RT. Are MDCK cells transfected with the
human MDR1 gene a good model of the human intestinal mucosa?
Pharm Res. 2002 Jun;19(6):765-72.
Regards
Varma Manthena
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