Hi all,
I am completely new in the field of Western Blot. I need antibodies directed against the bacterial gene "aac(6`)-Ib-cr" that codifies for a protein that is a fluroquinolone acetylating aminoglycoside acetyltransferase. I need to test the presence of this protein in my bacterium. I tried to have a look at the "Abcam" supplier, by searching by the name of the gene but I did not find anything. Is there another way to check if the supplier has these antibodies? (maybe the search by gene name is not suitable ?) Do you know other suppliers?What should I do if these antibodies are not commercially provided?Lastly, what kind of considerations should be done before choosing the most suitable antibody?
Thank you very much.
I would search PubMed or other libraries for similar work done by others, and check their materials and methods to see what they used. The more recent the papers, the better but even papers that were published more than 10 years ago can be helpful.
http://aac.asm.org/content/42/2/209.full
Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii
was published in 1998 and they used rabbit antisera, for one example.
You may have to make the antibody yourself, or have it made for you. You can either immunize with the entire purified protein (+ adjuvant), or identify a specific peptide for the enzyme that can be synthesized, conjugated to another protein such as KLH, and used as the antigen. There are many companies that will undertake this work for a fee and deliver an antiserum in a few months.
Thank you Adam.Could you please mention me some of these companies?Thank you
Dear
I recommend you to find some informations relative your question in this paper:
Salmonella enterica Serovar Typhimurium blaPER-1-Carrying Plasmid pSTI1 Encodes an Extended-Spectrum Aminoglycoside 6′-N-Acetyltransferase of Type Ib. Antimicrob Agents Chemother. 2003 February; 47(2): 697–703. Link: http://pubmedcentralcanada.ca/pmcc/articles/PMC151738/
Abstract: Almost all aac(6′)-Ib genes (synonymously called aacA4) described to date exist as gene cassettes carried by class 1 integrons. Preliminary resistance profile analysis suggested the presence of a single 6′-N-acetyltransferase. Besides being encoded by an integron-borne cassette and fused to the signal peptide of the OXA-1 β-lactamase, this acetyltransferase, called AAC(6′)-Ib11, was found to carry the novel amino acid combination of Leu118 and Ser119.
Hoping to be helpful
Marius
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