HiTrap® Protein G HP (Figure 3.10) is a convenient, ready-to-use column prepacked with Protein G Sepharose® High Performance. The columns are available in 1 mL and 5 mL sizes. In common with all HiTrap® columns, HiTrap® Protein G HP can be used for rapid antibody puriﬁcation with a syringe, pump, or chromatography system. Furthermore, puriﬁcation capacity can be greatly increased by connecting columns in series.
Figure 3.10.HiTrap® Protein G HP columns are designed for antibody puriﬁcation using a syringe, pump, or chromatography system.
Figure 3.11. shows the puriﬁcation of mouse monoclonal IgG1 on HiTrap® Protein G HP. The monoclonal antibody was puriﬁed from a hybridoma cell culture supernatant.
Figure 3.11.(A) Puriﬁcation of mouse monoclonal IgG1 from cell culture supernatant on HiTrap® Protein G HP 1 mL column. Purity of mouse IgG1 was conﬁrmed by (B) nonreducing SDS-PAGE on PhastSystem using PhastGel 10-15 (silver stained), and (C) agarose-gel immunodiffusion.
Refer to Chapter 2 (Desalting and buffer exchange) for general considerations.
The sample should be adjusted to the composition of the binding buffer. This can be done by either diluting the sample with binding buffer or by buffer exchange using HiTrap® Desalting, HiPrep 26/10 Desalting or PD-10 Desalting columns, Chapter 2 (Desalting and buffer exchange).
The sample should be fully solubilized. We recommend centifugation or ﬁltration immediately before loading on the column to remove particulate material (0.45 µM ﬁlter).
Never apply turbid solution to the column.
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.
For puriﬁcation using a syringe on HiTrap® Protein G HP 1 mL or 5 mL columns, buffers can be prepared from the 10× stock solutions of binding and elution buffers supplied with Ab Buffer Kit.
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.
* 1 mL/min corresponds to approximately 30 drops/min when using a syringe with a 1 mL HiTrap® column; 5 mL/min corresponds to approximately 120 drops/min when using a 5 mL HiTrap® column.
IgG from most species and subclasses bind to protein G at near physiological pH and ionic strength. For the optimum binding conditions for IgG from a particular species, consult the most recent literature.
Avoid excessive washing if the interaction between the antibody and ligand is weak, since this might decrease yield.
Protein G Sepharose chromatography media bind IgG over a wide pH range with strong afﬁnity at neutral pH. To elute the IgG, it is necessary to lower the pH to between 2.5 and 3.0 depending on the antibody. If biological activity of the antibody or antibody fragment is lost due to the low pH required for elution, try Protein A Sepharose; the elution conditions used are generally milder (Table 3.5).
Desalt and/or transfer puriﬁed IgG fractions to a suitable buffer using a desalting column (Chapter 2, Desalting and buffer exchange).
To increase capacity, connect several HiTrap® Protein G HP columns (1 mL or 5 mL) in series. HiTrap® columns can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system (Chapter 5 for details of ÄKTA chromatography systems).
Reuse of HiTrap® Protein G Sepharose HP columns depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.
Before storage we recommend to wash the column with 5 column volumes of 20% ethanol to prevent microbial growth. Store the column in 20% ethanol at 2 °C to 8 °C.