Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 207)

Fig. 1. Typical Chromatogram and SDS Page for Bio-Rad 1 ml eXact plus 10 ml Desalting Method*.
Profinity eXact affinity-tagged maltose binding protein (MBP) was purified from bacterial lysate by Profinia
chromatography using the preset affinity plus desalting method and recommended buffers. A Chromatogram of a
Profinity eXact purification using a 1 ml Bio-Scale Mini™ Profinity eXact cartridge and a 10 ml Bio-Scale™ Mini
Bio-Gel
® P6 desalting cartridge. B Samples were run on a 4–20% Criterion™ Tris-HCl gel. Lanes 1, Precision
Plus Protein™ standards (PPstd); lane 2, column load (Load); lane 3, column flow-through fraction (1A); lane 4,
column wash (1B); and lane 5, eluted and desalted tag-free MBP (1D).
Section 3
Frequently Asked Questions and Troubleshooting
Problem Possible Cause Possible Solutions
Protein detected in 1A Cartridge binding capacity Load a smaller volume of starting
(or 2A) flow-through fractions exceeded material or increase cartridge
size so as to not exceed binding
capacity
Sample or equilibration buffer Dilute sample with additional
not at optimum pH or contains binding buffer or adjust pH to <7
triggering anions such as CI - to ensure optimum sample pH;
remove triggering anions from
buffers used to prepare cell
lysate and wash buffer
Protein is sensitive to Maintain lysates at 4°C prior to
` temperature loading and run purification in
cold room or pre-chill buffers
No protein detected in 1D (or Sample does not contain target Check starting sample on
2D) elution fraction protein or target protein does SDS-PAGE to ensure that
not have affinity tag needed target protein is present and
for eXact system target protein (with affinity tag) is
intact
Protein lacks function in Protein is denatured or Modify buffer conditions and use
downstream assay or insoluble or is unstable in integrated desalting method to
appears degraded on elution buffer for long periods ensure quick buffer exchange
SDS-PAGE of time into a stabilizing buffer,
Protein is sensitive to Carry out purification at 4°C and
temperature set Profinia instrument to
cold-room operation setting
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A B
1 2 3 4 5