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Hi, I don't known if this list if the right place to ask for this
problem but I don't known other place
I am performing a tryptic peptide map of a monoclonal antibody
MoAb) in the following conditions :
Reduction & Alkylation of the MoAb in buffer Tris-HCL 100 mM with 10
mM DTT by incubation by 1 hr at 56 C and addition of Iodoacetamide at
55 mM ( final concentration) and incubation for 45 minutes in the
dark.
Digestion of the MoAb R&A with Trypsin sequencing grade (Boehringer
Mannheim) 1:10 - 1:20 ratio and incubation for about 18 hr at 37 C
This digestion, controled by SDS-PAGE look OK, almost complet
digestion of the protein,( I also check that I have the protein R&A
by SDS-PAGE, before I start the digestion, so I am not loosing the
protein in the eppendorf tube) but when I go to the chromatograpic
separation in RP-HPLC ( TSK-ODS , 5um RP-18 Pharmacia-LKB) with a
gradient of 0-100%B ( where buffer A is 0.1% TFA/H2O and buffer is
0.1% TFA/ACN )in 65 min. I just get a quit simple patern ( just one
peak in the first part of the gradient and the rest :blank ) I've
done this several time.
I am wondering if I am loosing my peptides in the eppendorf tube by
precipitation/insolubilization in the aqueous buffer.
Does anyone have any suggestions for this problem ?
Advices , protocols and ideas are very wellcome
Thanks in Advance
Lobvi E. Matamoros Fernandez
Protein Chemistry Lab
Center of Molecular Immunology
