Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum

Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum (http://www.molecularstation.com/forum/)
-   Proteomics Forum (http://www.molecularstation.com/forum/proteomics-forum/)
-   -   Identification of Antigen/Antibody of interest from vaccine (http://www.molecularstation.com/forum/proteomics-forum/416-identification-antigen-antibody-interest-vaccine.html)

minhphd 09-19-2006 02:53 AM

Identification of Antigen/Antibody of interest from vaccine
 
Hello every body.

I am a new member. My name is Minh. I come from proteomics Lab, Institute of Biotechnology, Hanoi, Viet nam.

I have some difficulties in a proteomics application to identify HBs Ag (from a recombinant vaccine sample). The sample comprises: HBs Ag 20ug/ml; Al(OH)3 < 1.25mg/ml; Themerosal< 0.1<%; formaldehyde<0.01%.
I tried several methods:
1. SDS-PAGE of 100ul sample (dryness), 1 observed band (Mw appro. 22kda) then was cut and followed by ingel-digestion and ESI-1DLCMS/MS identification (AB_QSTAR XL system).
2. Direct trypsin digestion (insolution digestion) of 100ul sample (dryness), following by ESI-1DLCMS/MS identification.
I spent more than 2 weeks on doing this work. now, I still do not identify the protein of interest.
I think that Al(OH)3 may impact on ability of trypsin digestion. But I don't know how to remove the impact.

If you have experiences or any idea, please suggest me how to solve this problem.
I am looking forward to hearing from you soon.

Thanks and best regards.

Pham Dinh Minh
Protein Biochemistry Lab, Institute of Biotechnology
Vietnamese Academy of Science & Technology

18 Hoang Quoc Viet Street, Cau Giay Dist., Ha Noi , Viet Nam

Tel: (+84)-4-7561903 Fax: (+84)-4-8363144 Emai: [Only registered and activated users can see links. Click Here To Register...]

oBWhat 09-19-2006 05:30 AM

Re: Identification of Antigen/Antibody of interest from vaccine
 
Dear minhphd,
welcome! :)

There are several solutions to this.

1) let the facility do the in gel digestion. Just give them your gel / band of interest.

2) You could dialyze the sample in dialysis tubing for a few hours with 50mM of Ammonium Bicarbonate or even easier use MINI-SLYDE a Lyzer dialysis tubes from Pierce and again diaylze with 50mM of Ammonium Bicarbonate.

Check with your mass spectrometry facility to see what they would like the samples in.

cheers and welcome to the forum! :)

minhphd 09-20-2006 08:14 AM

Re: Identification of Antigen/Antibody of interest from vaccine
 
Dear oBWhat

Thank you very much for the suggestions.
I tried some methods like that, such as recovery of proteins form ziptip c18, or ingel digestion. But the problem is I dont understand the bounds between proteins and AL(0H)3, and how the bounds impact on trypsin digestion and molecular weigh of peptides after digestion.

Please share me your ideas.

To all members in forum: If you have any ideas, please suggest me! Thanks alot!


All times are GMT. The time now is 10:51 PM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved

Page generated in 0.09226 seconds with 11 queries