Yes one word of warning about Simon's message below. If you compare mouse and rat sequences you will find that the isotypes (subclasses) match up as three are similar with three, with, in each species a duplication of a different one of the three giving rise to the fourth subclass (see Bruggemann reference for detailed explanation). A further complication arises in that some alleles of mouse IgG2a look as if they have arisen by an unequal crossover of two different mouse subclasses. What this all means in practice is that if a polyclonal reagent is adorbed against one species (e.g. mouse IgG)it can render the reagent as behaving as subclass biased against the other species (rat IgG). Cheers, Mike On May 29 2008, Simon Hunt wrote: >Hello Rudjer, > > I endorse Ray's recommendation of Southern Biotech's polyclonals, though > for 1-colour work antibodies specific for one isotype [or allotype] > shouldn't be necessary. So an "anti-IgG" antibody like the ones you > describe should be OK. > > I'm guessing, but are you doing 2-colour work, using a mouse monoclonal > as the primary for the other colour? Or are you using mouse cells which > may include some Ig(G) positive mouse B cells? If so, then you will you > have to be careful, since unadsorbed polyclonal antibodies used as > secondaries will cross-react between mouse and rat. Southern Biotech and > other reputable suppliers like AbD-Serotec > (http://www.antibodydirectory.com/html/abd/index.htm ) are well aware of > this and you can trust them to have removed the unwanted anti-mouse > component by absorption, to pick out just your primary rat Ig without > cross-reaction. > > Alternatively you can use conjugated mouse anti-rat monoclonals as > secondaries, where the species cross-reactivity problem obviously won't > arise. There are lots of these in the catalogues, specific for the > particular isotype you want. > > I think you do mean isotype or perhaps subtype, not allotype, in your > question. Allotypes refer to the within-species genetic polymorphisms, > i.e. the differences between different rat strains. Although there are > well-described allotype-related sequence variants in rat Heavy chains, > they are very unlikely to matter when choosing your second layer > antibody. > > Marianne Brüggemann and her colleagues sequenced the different rat Ig > heavy chain isotype genes in the late 1980s. You can either look up her > papers or go to the databases if you really want the sequences. I've just > run a job on UniProt called Z8GP which allows you to read and compare > [use "Align"] the Heavy chains of rat Igs: > http://beta.uniprot.org/uniprot/?query=job%3AZ8GP. There are multiple > primary sequence differences between the different isotypes, as you'd > expect. However, I don't think they will help you with choosing the right > antibody. If you want to get a feel for the different tertiary structures > of Igs visit Mike Clark's webpages on mouse and human Ig structures > (http://www.path.cam.ac.uk/~mrc7/mikeimages.html ). > >Good luck with your research, > >Simon Hunt > > >In message <000a01c8c022$93d092c0$bb71b840$@com> "Ray Hicks" ><rhicks@cytekdev.com> writes: >> Hi Rudjer, >> >> I've always found the Southern Biotechs' IgG-specific antibodies are >> highly specific and have minimal cross-reaction with other species or >> isotypes. I've never tried their anti-rat IgG2a, but they have it in a >> few useful conjugations: FITC,PE,Cy5 and Biotin (go to >> http://www.southernbiotech.com/cgi/productsearch.asp and search for >> "anti-rat Igg2a" (note that "AP" is alkaline phosphatase in their >> catalogue not APC, so not so useful for flow), >> >> Cheers >> >> Ray >> >> Ray Hicks >> Cytek Development Inc >> http://www.cytekdev.com >> tel: +44 (0)208 1337 968 (UK/Europe) >> fax: +44(0)208 5889 004 >> skype: ray.hicks.cytek >> >> >> -----Original Message----- >> From: Rudjer Novak [mailto:rnovak@pharma.hr] >> Sent: 26 May 2008 10:42 >> To: cyto-inbox >> Subject: IgG allotypes >> >> dear flowers, >> >> I use a primary (rat) IgG2a antibody and need a secondary, but I can't >> find an IgG2a specific one. The best I could find was a polyclonal anti >> rat IgG Ab and a H+L specific anti rat IgG Ab (which sound the same to >> me?). Is that gonna work? Would you prefer any of the mentioned >> secondaries? What is the structural difference between IgG allotypes? >> >> thanks for your time! >> rudjer >> >> ________________________ >> >> Rudjer Novak, dipl. ing. >> Farmaceutsko-biokemijski fakultet >> Zavod za biokemiju i molekularnu biologiju >> A, Kovacica 1 >> 10000 Zagreb >> tel: 095 804 3102 >> ________________________ >> >> Rudjer Novak, B.Sc. >> University of Zagreb >> Faculty of Pharmacy and Biochemistry >> A. Kovacica 1, Zagreb >> Croatia >> >> > -- M.R.Clark, PhD Reader in Therapeutic and Molecular Immunology University of Cambridge, Department of Pathology Tennis Court Road, Cambridge CB2 1QP Tel +44 1223 333705 Fax +44 1223 333875Received on Tue Jun 3 12:18:00 2008
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