[Immunology] tryptic peptide mapping

GennovaQC GennovaQC at emcure.co.in
Mon May 29 22:27:28 EST 2006


Hello,
 
I found your question about peptide mapping posted in as follows
 
Please let me know if you have received or found any answer to the same
problem. I am also facing the same problem.
 
Thanks

tryptic peptide mapping

Lobvi Matamoros LOBVI at ict.sld.cu
<mailto:immuno%40iubio.bio.indiana.edu?Subject=tryptic%20peptide%20mappi
ng&In-Reply-To=> 
Fri May 10 00:43:06 EST 1996 
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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
 
 
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