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Immunoaffinity Chromatography 59
absorbance must be used during coupling with NHS-activated supports. Vendors
who offer activated supports include GE (http://www.amershambioscienes.com),
Millipore (http://www.millipore.com), BioRad (http://www.biorad.com), JT Baker
(http://www.jtbaker.com), and Tosoh Bioscience (http://www.tosohbiosep.com).
3. NHS-activated Sepharose HP is only available in prepacked HiTrap columns;
however, NHS-activated Sepharose 4 Fast Flow is available as unpacked gel in a
range of resin volumes. The unpacked gel has the advantage of allowing packing
of a broad range of column sizes.
4. As NHS interferes with absorbance measurements near 280 nm, A 280 nm will not be
effective for determination of coupling efficiency.
5. A flow rate of between 1 and 5 ml/min (30–150 cm/h) may be applicable for a
2.5 cm (long) by 1.6 cm (in diameter) column of NHS-activated Sepharose HP.
This resin has an average diameter of 34 μm and should provide relatively low
pressure drop. However, excessive pressure should be avoided as this can damage
the packing of the column (normally the resin is not damaged as it is compressible).
6. If the load has previously been partially purified by some other means (ion exchange
chromatography, precipitation, and so on), the purification will be enhanced by
putting the load into a good loading buffer such as 10 mM HEPES, 500 mM NaCl,
0.01% Tween 80, pH 7. This can be accomplished by dialysis (described above) of
the load into the buffer or by dilution of the protein solution with the buffer until
the desired pH is reached (dilution should not be less than 1 part load to 2 parts
buffer). This prepared load should be 0.2 μm filtered and loaded onto the column
as described above.
7. Clarification to a final 0.2-μm filtration can be accomplished at small scale by
centrifugation followed by vacuum filtration with a glass fiber prefilter (provided
that the protein of interest does not bind to the glass prefilter). Centrifugation at
Gt=10 6 sec will remove cells and large cell debris. Small insoluble particles that
can foul the column will be removed by 0.2-μm filtration. If only filtration is to
be employed, the following filter train works quite well for most mammalian cell
culture supernatants: Sartorius Sartopure PP2 1.2-μm filter followed by a Sartopore
2 0.45/0.2-μm filter.
8. The amount of product to load depends on a number of factors, including protein
size, load composition, and chromatography resin. In this example, 0.03 mg of
target protein per ml of resin was loaded. Loading more than the capacity of the
column will result in loss of the product in the flow-through and wash.
References
1. Janeway, C. A., Travers, P., Walprot, M., and Shlomchik M.J. (2005) Immunobiology,
The Immune System in Health and Disease, pp 151. Garland Science,
New York.
2. Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, pp 53–244. Cold
Spring Harbor Laboratory, United States of America.