Protein G GraviTrap™ are gravity-ﬂow columns prepacked with 1 ml of Protein G Sepharose® 4 Fast Flow. The columns are designed for fast and efﬁcient manual puriﬁcation of monoclonal and polyclonal antibodies and antibody fragments from cell culture supernatant and biological ﬂuids. The antibodies are simply captured with high speciﬁcity on protein G ligands in the gravity-ﬂow columns. You do not need any other instrument with this protocol because the entire process relies on the ﬂow of gravity. Together with the packaging that can be converted into a column stand (Workmate), parallel puriﬁcation is made simple (Figure 3.4). The columns are reusable up to ﬁve times.
Figure 3.4Protein G GraviTrap™ are prepacked gravity-ﬂow columns that provide simple, manual puriﬁcation of antibodies from cell culture.
rProtein A Sepharose® and a mix of Protein G Sepharose® 4 Fast Flow and rProtein A Separose® 4 Fast Flow are also available in GraviTrap™ format. The simple four-step procedure for puriﬁcation is shown in Figure 3.5.
Figure 3.5Purifying antibodies with Protein G GraviTrap™ is a simple four-step procedure.
Refer to Desalting and buffer exchange for general considerations.
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 (see 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.
Buffers can be prepared from the 10× stock solutions of binding and elution buffers supplied with Ab Buffer Kit (28903059).
As a safety measure to preserve the activity of acid-labile IgG, addition of 1 M Tris-HCl, pH 9.0 to the tubes used for collecting antibody-containing fractions (60 to 200 µl/ml eluted fraction) is recommended. In this way, the ﬁnal pH of the sample will be approximately neutral.
The eluted fractions can be buffer exchanged using PD-10 Desalting columns or HiTrap Desalting columns (see Desalting and buffer exchange).
Depending on the nature of the sample, Protein G GraviTrap™ columns may be reused up to ﬁve times consecutively. Reuse of the columns should only be considered when processing identical samples to avoid cross-contamination.