Reductive Cross-linking of Proteins to Antibodies
This procedure is useful for site-specific conjugation of proteins (such
as the phycobiliproteins PE and APC, as well as their tandems) to antibodies.
It is used in our antibody conjugation protocols for all phycobiliprotein-based
fluorescent dyes, as well as conjugation of Texas Red BSA to antibodies.
Finally, a version of it is also used to conjugate these fluorescent proteins
to non-immunoglobulin proteins (such as Annexin V).
The concept of the conjugation is simple: Derivatize the fluorescent protein
such that it has sulfhydryl-reactive maleimide group, and then reduce the
hinge disulfide on the immunoglobulin to provide the reactive site. The
first derivatization is accomplished using the heterobifunctional cross-linking
reagent "SMCC". This molecule has two ends: one reacts with primary
amines; the other end is a maleimide group that will react with free sulfhydryls.
SMCC is conjugated to the fluorescent proteins through simple amino-reactive
conditions (i.e., basic pH), such that a few molecules are covalently conjugated
to each protein. This derivatized protein is purifed away from the free
SMCC. It is actually stable for extended periods of time; we have stored
SMCC-conjugated phycobiliproteins in the MES buffer for several months in
the refrigerator and then mixed them with the reduced immunoglobulins--with
no notable loss in activity.
The immunoglobulin is treated with DTT to expose free sulfhydryls, and exchanged
over a column to remove free DTT. Probably only one of the suflhydryls
is then derivatized by the SMCC-protein conjugate (due to steric hindrance).
The real advantages of this protocol thus becomes apparent: the conjugation
is well-defined, and the conjugation does not interfere with the binding
site of the antibody.
After the covalent coupling of the SMCC-protein to the immunoglobulin, NEM
is added to block any more free sulfhydryls (on the immunoglobulin). Then,
the reaction mixture is exchanged onto storage buffer, which also removes
free NEM. Note that the final reagent can have unconjugated fluorescent
proteins (e.g., unconjugated PE). Normally, this protein washes off after
the cell-staining step. In case of excessive background or in conditions
where washing may not be performed, it may be desirable to purify the product
using Protein A or Protein G columns. Fractionation by sizing can be difficult,
since the combined molecular weight (e.g.) of an IgG-PE conjugate is 400,000,
compared to 240,000 for free PE.
Interestingly, many IgM's can also be derivatized by this protocol; each
particular antibody should be conjugated by this protocol and tested for