Protein G Sepharose® 4 Fast Flow consists of 90 µm beads of highly cross-linked agarose, which provide a robust and stable chromatography matrix that allows high flow rates. The medium is a good choice for general-purpose capture of antibodies and scale-up in the laboratory.
Protein G Sepharose® 4 Fast Flow is available as a bulk medium (Figure 3.2) for packing in XK, HiScale, and Tricorn columns (Figure 3.3) at laboratory scale. Furthermore, Protein G Sepharose® 4 Fast Flow can be packed in larger columns for industrial-scale purification of monoclonal and polyclonal antibodies.
Figure 3.2.Protein G Sepharose® 4 Fast Flow is available in various bulk (lab packs) sizes for laboratory and process-scale applications.
Refer to Column packing and preparation for general column packing guidelines.
Sepharose® 4 Fast Flow chromatography media should be packed in XK or Tricorn columns in a two-step procedure: Do not exceed 0.3 bar (0.03 MPa) in the ﬁrst step and 3.5 bar (0.35 MPa) in the second step. If the packing equipment does not include a pressure gauge, use a packing ﬂow rate of 2.5 mL/min (XK 16/20 column) or 1.0 mL/min (Tricorn 10/100 column) in the ﬁrst step, and 14 mL/min (XK 16/20 column) or 5.5 mL/min (Tricorn 10/100 column) in the second step.
Figure 3.3.Empty Tricorn columns for packing. Once packed, these columns can be used for puriﬁcation using either a pump or chromatography system.
For subsequent chromatography procedures, do not exceed 75% of the packing ﬂow rate.
Centrifuge samples (10 000 × g for 10 min) to remove cells and debris. Filter through a 0.45 µM ﬁlter.
The sample should have a pH around 7.0 before applying to the column. If required, adjust sample conditions to the pH and ionic strength of the binding buffer by either buffer exchange on a desalting column (Desalting and buffer exchange) or dilution and pH adjustment.
Binding buffer: 0.02 M sodium phosphate, pH 7.0
Elution buffer: 0.1 M glycine-HCl, pH 2.7
Neutralizing buffer: 1 M Tris-HCl, pH 9.0
Water and chemicals used for buffer preparation should be of high purity.
Filter buffers through a 0.45 µM ﬁlter before use.
To preserve the activity of acid-labile IgG, we recommend adding 60 to 200 µL of 1 M Tris-HCl pH 9.0 to collection tubes, which ensures that the ﬁnal pH of the sample will be approximately neutral.
Appendix 7 for information on how to convert linear ﬂow (cm/h) to volumetric ﬂow rates (mL/min).
Desalt and/or transfer puriﬁed IgG fractions to a suitable buffer using a desalting column (Desalting and buffer exchange).
Store in 20% ethanol at 2 °C to 8 °C.
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