View - ResearchGate

58 Gallant et al.
Fig. 2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
and Western blot of immunoaffinity eluate with MW standards (lane 1), reference
protein (lane 2), immunoaffinity eluate (lane 3). (A) SDS–PAGE gel stained with
Coomassie Blue, (B) anti-target Western blot, and (C) anti-host cell Western blot.
and Western blots seen in Fig. 2. Only a small amount of host cell protein
remains as a contaminant of the immunoaffinity-purified protein.
4. Notes
1. A large number of companies provide these services. Two that the authors
have found to be reliable and economical are Covance (http://www.covance.com)
and Sierra Biosource (now Celliance, http://www.celliancecorp.com). Some
approximate times for production for polyclonal sera is 3 months and for monoclonal
hybridoma development is 6 months.
2. Some considerations when selecting the activated support include that the pore
size should be adequate for the antibody and the protein of interest. Because a
layer of bound antibody extends approximately 10 nm away from the pore wall,
antibody immobilization can significantly narrow a 50-nm pore. Relatively, large
pore-activated supports (in the range of 100–300 nm) are available through Millipore
in their Prosep line of activated supports; Millipore has aldehyde activated controlled
pore glass, as well as glyceryl CPG that must be oxidized to the aldehyde form.
Coupling through the lysines with an aldehyde-activated support is a particularly
effective means of covalently attaching antibodies. Evaluation of several different
methods of coupling for coupling efficiency is useful at the beginning of a project.
Measuring coupling efficiency through UV absorbance is only appropriate if the
coupling reaction does not release a UV-absorbent product. For example. NHS
chemistry releases a UV-absorbent NHS group for each covalent bond formed
during coupling, so another method of protein concentration determination than UV