Bio-Rad Rotofor® and Mini Rotofor Cells - Manual (Page 27)

2. Temperature. Many proteins, especially enzymes, are temperature labile. Make
sure that the water recirculation chiller is set 10°C cooler than the temperature
required to maintain stability of your protein. The heat generated during IEF
keeps the temperature inside the focusing chamber approximately 10°C higher
than that of the circulating coolant. Temperature settings for chillers are generally
between - 10°C and 4°C.
Diffusion rates of proteins are directly proportional to their temperature.
Because proteins at steady state diffuse in and out of their focused zones it is
advisable to run the Rotofor cell at the lowest possible temperature to offset
this effect.
3. Ampholytes. Ampholytes may form weak electrostatic complexes with proteins.
They can be removed by bringing pooled fraction(s) to 1.0 M NaCl and dialyzing
against appropriate buffer or water. The salt effectively exchanges for the
ampholytes on the protein. This may be followed by dialyzing against appropriate
assay buffer. Other methods for ampholyte removal include electrophoresis;
ammonium sulfate precipitation; and gel filtration, ion-exchange, and hydroxylapatite
chromatography. Be sure to measure the pH of the fractions before manipulating
them to remove ampholytes.
4. Urea. Urea in the focusing media at 3 M generally alleviates precipitation.
Without the use of urea, loss of activity due to precipitation may be excessive.
Urea at higher concentrations (4-8 M) is often used. Following focusing, dialysis
will remove urea from the solution.
5. Detergents. Both the concentration and the type of detergent used play an
important role in recovery of activity. Use the least amount of compatible detergent
required to maintain the solubility of your protein. Also try other non-ionic or
zwitterionic detergents. Removal of detergent from Rotofor fractions may be
necessary for full recovery of activity.
6. Precipitation. Protein-protein interactions may result in activity loss.
Decreasing the protein load, and addition of detergents, glycerol, reducing
agents and/or chaotropic agents keep proteins from forming complexes during
focusing.
7. Proteins are not always active at their pI. Adjust the pH of the solution for
assay.
8. Some proteins require the presence of a particular ionic species for activity
(i.e. mono- or divalent cations like Na + or Mg
2
+ ). Replace the ions, if necessary,
for assay.
9.4 Maximizing Resolution
1. Diffuse or multiband protein IEF patterns can arise from molecular interactions
and conformational changes as well as from inherent isoelectric microheterogene-
ity. Ampholytes can reversibly bind to proteins, proteins can undergo sequential
pH dependent conformational changes, and proteins can interact with one anoth-
er. These types of reactions can artifactually alter the pH profiles of proteins. On
the other hand, many proteins are inherently heterogeneous, consisting of isoelec-
tric isomers. To distinguish between artifactual and inherent heterogeneity, it may
be necessary to run an analytical IEF gel in the presence of all constituents to be
used during focusing in the Rotofor cell (i.e., detergents, urea, glycerol, etc.) in the
same proportions to be used in the Rotofor cell. Single focused bands should be
cut out and rerun. If this single band splits into many bands, artifact formation is
indicated. In this case the Rotofor “prefocusing” protocol is recommended.
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