Bio-Rad Profinia™ Protein Purification Instrument User 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
200
A
B
1 2 3 4 5