only be performed where the epitope is linear. Alinear epitopeis formed by amino
acids lying adjacent to each other and the antibody binds to the structure that they
form. Non-linear epitopes are formed from non-adjacent amino acids when
they interact with each other in space, as is found in helical or hairpin structures.
To carry out epitope mapping the amino acid sequence of the target protein must be
known. The sequence is then used to design and makesynthetic peptideseach
containing around 15 amino acid residues in length and overlapping with the pre-
vious one by about five residues. The synthetic peptides are then coated on to the wells
of microtitre plates or onto nitrocellulose membranes and reacted with the antibody of
interest. The reaction is visualised by using a secondary antibody enzyme conjugate
and substrate. From the reaction to the peptides and the position of the sequence in the
native protein it is possible to predict where the epitope lies and also what its sequence is.
7.7 Immunoblotting
This technique is also known aswestern blottingand is used to identify proteins from
samples after electrophoresis. The sample may be tissue homogenate in origin or an
extract of cells or other biological source. The sample may be electrophoresed under
reducingornon-reducingconditions until separation is achieved. This is usually
visualised by staining with a general protein stain. The separated proteins are trans-
ferred onto a nitrocellulose or polyvinyl membrane either passively or by using an
electroblotter. The membrane is treated with a protein-blocking solution to prevent
non-specific binding of antibody to the membrane itself. Popular blocking com-
pounds are dried milk or bovine serum albumin. Either direct or indirect antibody
systems can be used but often indirect methods are used for reasons of cost. Directly
conjugating primary antibodies may be expensive and so very often anti-species
enzyme conjugate is used. For indirect labelling the membrane is incubated in
antibody solution and after washing, it is treated with a solution of a secondary
antibody–enzyme conjugate. Both peroxidase and alkaline phosphatase have sub-
strates that will produce a solid colour reaction on the blot where the antibodies have
bound. The substrate reaction can be stopped after optimum colour development,
dried and the blots stored for reference. Further details can be found in Section 10.3.8.
The method is particularly useful during development of new antibodies as part of
epitope mapping studies.
7.8 Fluorescent activated cell sorting (FACS)
Fluorescent activated cell sorting (FACS) machines are devices that are capable of
separating populations of cells into groups of cells with similar characteristics based
on antibody binding (Fig. 7.17). The technique is used on live cells and allows recovery
and subsequent culture of the cells after separation. Many cell markers are known
which identify subsets of cell types and specific antibodies to them are available.
293 7.8 Fluorescent activated cell sorting (FACS)
