Three types of starting materials are available for use as antigens, including purified native proteins, bacterial fusion proteins or short synthetic peptides.
In our laboratory we have emphasised the use of short peptides as they allow the targeting of specific regions of a transporter. This is particularly important when the transporter may be alternatively spliced or shows significant sequence homologies to other members of a family of transporters.

Short peptide sequences can be chosen with reference to databases such as Genbank, that are specific only to a single member of a family of transporters, and thus have the potential to be monospecific. Additional structural modifications such as phosphorylation can also be included in the design of the peptide. Peptides must be chemically coupled to carrier proteins in order to render them immunogenic, and in some cases individual sequences may not be recognised as foreign by the immune system of the animal that is immunised. The basic rules detailing peptide selection are presented below.

Peptide Length:
Peptides that are 9 residues or shorter may sometimes be effective antigens; conversely, peptides longer than around 16 amino acids may contain several epitopes. Most workers will obtain peptides of 10-15 amino acids, synthesised commercially, with relevant quality control data appended.

Where to Start:
Computerised tools exist for identifying unique and appropriate sequences of transporters including features such as uniqueness of the sequence and potential antigenicity.

Sequence Databases:
Databases such as the GenBank database run by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) provide a sensible starting point for comparing the sequences of cloned transporters from a variety of species. Selection of a sequence may be driven by a need to use the antibody in one or more species, in which case the sequences of different species must be evaluated to ensure significant similarity in the chosen region. Use of the BLAST algorithm for comparing short sequences ( http://www.ncbi.nlm.nih.gov/BLAST/ ) is strongly recommended. Conversely the Conserved Domain and Search Service (http://www.ncbi.nlm.nih.gov/Structure/ ) allows identification of common structural domains such as PDZ domains, phosphorylation consensus sites etc. The common nature of such sequences will influence your usage of such.

Intracellular, Extracellular and Transmembrane Sequences:
It is easier to produce antibodies against intracellular domains of proteins as there is no possibility that clonal elimination by the immune system has occurred (a normal mechanism to prevent the animal recognising self-proteins. Intracellular sequences are not normally exposed to prior surveillance by the immune system and are thus usually antigenic. Alternatively, if a protein is sufficiently variable between species it may be raised in a remote species without encountering this ‘self’ identification barrier.

Transmembrane sequences:
We don’t try to raise antibodies to these regions as they are usually hydrophobic and buried in the lipid membrane where they are not accessible to antibodies in techniques such as immunocytochemistry. Many databases containing cloned sequences often contain suggestions as to the likely membrane topology of a protein. This assignment may be based upon a variety of experimental and inferential data, but may be supplemented by the use of computational techniques such as Kyte-Doolittle hydropathicity plots which try to predict potential transmembrane regions based on the presence of hydrophobic regions. A variety of programs are freely available on the Web. (eg see http://fasta.bioch.virginia.edu/o_fasta/grease.htm). Whilst this is helpful it is not an unequivocal tool, since a transmembrane domain may in fact contain many hydrophilic amino acids if it forms a pore complex. Conversely, some hydrophobic residues may exist in an aqueous environment. However it is unwise to choose a hydrophobic region as an antigen since hydrophobic peptides are difficult to dissolve in aqueous solvents, for coupling to a carrier protein.

Measuring ‘Antigenicity’:
A variety of tools can calculate the probability that a given portion of a protein sequence may be antigenic. The antigenicity index is calculated using multiple measures of secondary structure, including hydrophobicity, flexibility and presence of strong turns or coils. A variety of free programs can be accessed on the Web.
Further choice of peptide sequences also needs to consider whether it contains one or more potential post-translational modification sites such as glycosylation site(s) or phosphorylation sites, which could render the resultant antibody specific only to the non-glycosylated forms of the protein etc.
Modifications such as phosphorylation sites can potentially be predicted using programs such as Netphos, ( http://www.cbs.dtu.dk/services/NetPhos/).

Cleavage Sites:
Many proteins are proteolytically cleaved during processes such as bio-activation or degradation. Web based tools can identify possible cleavage sites, eg. see http://au.expasy.org/tools/peptidecutter/.

Purity of the Peptide:
Peptide impurities are usually of three sorts. Shortened (truncated) peptides are not normally problematic, because they have the correct amino acid sequence but are shorter. Mis-sequences (peptides with missing residues) may ultimately result in production of antibodies which recognize inappropriate proteins. Finally, if amino acids are not fully de-protected then they tend to be more immunogenic than the native peptide (possibly because they are more resistant to degredation) BUT, antibodies directed against the modified peptide may not recognize the native peptide.
If cost is a concern it may be appropriate to initially immunise an animal with cheaper grade peptides. If an immune response is generated, then a small amount of more expensive pure peptide can be used for further immunisation. However, in general, it is recommended that when all costs are taken into account, the use of purest quality peptides from the outset may be the most cost-effective and reliable route to take.
Peptides are offered with either free amino and carboxyl terminal regions or with these regions modified. It is common practise to acetylate the N-terminal as this may facilitate proper folding of peptide sequences. Other modifications may be included to aid coupling of the peptide to the carrier protein, including addition of a terminal –SH-containing amino acid such as cystine. Phosphopeptides can be readily synthesised in a relatively pure form and may be appropriate when studying the phosphorylation status of proteins. Most commercial suppliers can include a phosphorylated residue in the synthesis of the peptide of interest. As the phosphate is to be a significant component on the epitope, the length of the peptide should be relatively short (10-14 amino acids) to ensure it is a part of the epitope that the antibodies will recognise.

Carrier Protein:
Carrier “proteins” are large molecular backbones that the small peptide antigens are attached to, thus rendering them immunogenic (able to render an immune response). We routinely use porcine thyroglobulin for most of our work
but other such as keyhole limpet haemocyanin, or synthetic polylysine etc supports (MAPs) can be used Some MAPS are also available with modified groups which enable them to be chemically coupled to peptide sulphydryl groups, for use in circumstances where the peptide was not synthesised as part of the MAP.

Raising Antisera:
The peptide needs to be conjugated to a suitable carrier in order to render it immunogenic. We routinely couple at the ratio of 1 mg of a 20 amino acid peptide per 25 mg of thyroglobulin. This ratio is limited by the number of lysine residues present on the carrier protein. The weight of peptide used should also be changed in a pro-rata fashion as the length of the peptide changes (eg. use 0.5 mg of a 10 amino acid peptide).

Coupling via an Amino Group:
This is the simplest method. We describe other methods in Pow et al., (2003). Two types of amino group may exist in a peptide: an N-terminal amino group and the free -epsilon amino groups of internal lysine residues.
Crosslinking reactions using aldehydes preferentially react with the epsilon-amino group on the lysine residues, causing the peptide to be stably coupled to the
carrier at that part of its sequence. If you are targetting an amino terminus region of a protein, it is unwise to couple up the amino terminal region of the peptide as this will make it structurally distinct from the comparable region of the native protein.
This discussion assumes the use of rabbits as host animals. Before immunising any animal, take a test bleed and utilise the serum in the type of test system you will employ it in. It is extremely common for some animals, especially those that are not reared in specific pathogen free conditions, to have pre-existing antibodies which give rise to high background levels or non-selective labelling of tissue constituents when used in immunocytochemical studies. Screening and elimination of those animals with unacceptable levels of contaminating antibodies at this point will save considerable work at later time points. The retained serum sample will serve as a useful immunocytochemical control at a later time.
For specific conjugation/immunisation protocols, see Pow et al., 2003.

Methods to test the specificity of the antibody:
A variety of methods are normally employed to determine if you have any antibody that can detect the target sequence, and whether it solely detects the target protein. These include dot blots with the target peptide, western blotting, and strategies such as pre-absorption of antibodies with target peptides. We discuss these in more detail in Pow et al., 2003. Using the Antibodies for Immunocytochemical Studies
Only when you are certain that you have a specific antibody should you embark on immunocytochemical studies. Most transporters require the tissue to be fixed with paraformaldehyde rather than glutaraldehyde. In the first instance, fixation may have to be varied from relatively short mild fixation, up to 4% paraformaldehyde for 1-2 hours. Our postmortem human tissue studies may use tissues fixed for 24 hours or more.
We routinely use paraffin-wax-embedded tissues for our immunocytochemical studies. These will always require antigen recovery. We use the Revealit antigen recovery solution (but note they currently sponsor our web site so this is not entirely unbiased, but it works well!!!).

Colocalisation of Transporters, Amino Acid Neurotransmitters and Other Related Molecules:
We frequently utilise immunocytochemistry to localise not only the transporter but also the molecule that the transporter is thought to translocate. This is particularly important when the transporter may have multiple potential substrates and the investigator wishes to determine if the cell type has an enriched content of one specific molecule. We utilise antibodies which have been specifically created against paraformaldehyde conjugates of amino acids, and which thus recognise these amino acids in paraformaldehyde-fixed tissues. By localising the multiple components of biological systems such as amino acid neurotransmission systems it is possible to better understand the functioning of the whole system.
We hope this is of assistance and are happy to answer specific queries. Good luck!