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Dear technical team,
Our customer is going to purchase ab30522.
She will test this antibody on gold surface chip which is bounded protein A.
She needs to detect using FITC so choose ab30522 but concerns if binding site of protein A and FITC conjugated site are overlapped.
If you have information, please let me know.
Asked on Apr 30 2012
Thank you for your enquiry.
It might be possible for a FITC-conjugated IgG to still be able to bind to Protein A as well, primarily due to the fact that FITC conjugation to IgG and Protein A binding to IgG occur at different sites within the IgG molecule and utilize different modes of attachment. FITC binds to IgG via primary amines which are distributed in both the Fab and Fc regions. On the other hand, Protein A binding to IgG is confined to the Fc region; this non-covalent interaction involves the participation of carbohydrate (oligosaccharides) moieties attached to particular amino acids (e.g Asn) in the Fc region and the amino acid residues of Protein A.
Based on these considerations, it ispossible for IgG-FITC to still be conjugated to Protein A. However, there is also the bulk of the molecules to consider. Wedo not have an exact ratio of FITC to IgG (typical ratio will be 3 to 10) and we do not know exactly which lysine residues are conjugated, or more importantly, how many of the ˜15 lysine residues in the Fc region of rabbit IgG are conjugated to FITC, which can possibly contribute some steiric hindrance effects and affect binding of the Protein A to the Fc region. One Protein A molecule is typically able to bind two or more unconjugated IgG molecules; bulkier IgG may reduce this binding.
I hope this information will be helpful. Please note that the E. coli antibody (FITC) antibody has regrettably not been tested and guaranteed for this particular application that the customer would like to use.
If you have any further questions, please do not hesitate to contact us.
Answered on Apr 30 2012