Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 199)

Fig. 1. Typical chromatogram and SDS-PAGE for Bio-Rad 1 ml Protein A plus 10 ml desalting method.
Section 3
Frequently Asked Questions and Troubleshooting
Problem Possible Cause Possible Solutions
Antibody detected in 1A Cartridge binding capacity Load a smaller volume of starting
(or 2A) flow-through fractions exceeded material or increase cartridge
size so that the binding capacity
is not exceeded
Sample or equilibration buffer Dilute sample with additional
not at neutral pH or correct ionic binding buffer or adjust pH to
strength for optimum binding
ensure neutral sample pH
Sample loading is too fast for Choose the “Low” flow rate
` efficient binding option in the Protein A methods
and, if necessary, dilute sample
to reduced concentration of IgG
No antibody detected in 1D Sample does not contain Check starting sample on
(or 2D) elution fraction immunoglobulin or SDS-PAGE to ensure that
species/subclass of IgG does antibody is present and refer to
not have affinity for Protein A cartridge manual to confirm
specificity of species/subclasses
for Protein A resin
Antibody lacks function in Antibody is acid labile Increase pH of elution buffer and
downstream assay or use Protein A Custom method to
appears degraded on collect larger volume from elution
SDS-PAGE fraction
Antibody is temperature Carry out purification at 4°C and
sensitive set Profinia instrument to
cold-room operation setting
Low purity of antibody on Sample load and wash steps Select “Extended” wash option
SDS-PAGE or residual do not return to baseline with for Protein A method during setup
contamination from standard Protein A method to increase wash time
starting material wash option
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Fraction 1A-Flow-through, 1B-Wash, 1D-Affinity-purified + Desalted IgG