as a routine, when we characterize a new phosphorylation site, we make the
phospho and the dephospho peptide, raise antibodies, clear the serum with
an affinity column of the dephosphopeptide and pull out the antibody on a
phosphopepetide affinity column. We have been able to use these very
specific antibodies to demonstrate in-vivo phosphorylation events involving
phosphorylation at different sites on the same protein under different
stimuli, all by simple western blots, probably the easiest and most
specific way to analyze in-vivo phosphorylation in a site specific manner.
Just treat your phosphopeptide antigens as per a normal peptide antigen,
design the peptide with the phosphate somewhere in the middle, we usually
make 12 to 15mers with the phosphate at 6 or 7, split the resin and add a
cysteine at the N-terminus of half the resin, cleave, couple the reduced
peptide to KLH-SPDP and off you go!
Phosphopeptide/KLH conjugates make very good antigens.
Good luck....Ken
>We don't have any experience using phospho peptides for antibodies. Is it
>recommended?
>
>Thanks for the help as usual,
>
>Lisa
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Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
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