While concentrating the sample, nucleic acids can be removed and the target protein can be enriched to a certain extent. We have found a remarkable resin, Affi-GelBlue, which has been used as an affinity chromatography resin for the purification of hundreds of proteins over the years, primarily in subsequent stages of the purification step (see Bio-Rad Bulletin 1107, biorad.com). By this method, some of the target proteins can bind to the resin , and can be eluted with gradient salts or specific ligands. This chapter will focus on the use of Affi-Gel Blue in the early procedures of purification, generally in the form of direct mixing for purification purposes. Authors: Burgess et al, Translator: Chen Wei, This experiment is from "Protein Purification Guide".
Operation method
Classic program Move I. Typical programs Sample the supernatant components at either the low (S10) or high (S100) rate. Sampling at the high rate is generally preferred, as it eliminates interference from ribosomes or microsomes. The size of the column used depends on the sample volume. A useful rule of thumb is to use I mL of packed gel (50 to 100 mesh) for every 100 m g of protein in the sample. According to the manufacturer (Bio-Rad, Bulletin 1107), I mL of filled Affi-Gel Blue binds 11 m g of protein. In general, only 5% to 10% of the total protein can be bonded on the column under these conditions, so the protein to gel ratio suggested here is sufficient. However, for each extract, a specific analysis of the flow-through material should be used to ensure that the target protein is completely bound to the column. If this analysis is not possible, the following should be used. A buffer has been found to be effective as a starting buffer for chromatographic analysis, together with an appropriate stabilizer, and is formulated as 10 m m o l / L Tris-H C l with 0.1 m o l / L K C l , p H 7.5. The sample should be suitable for 0-I m o l / L K C 1 . The sample should be adapted to 0-I m o l/L Kcal 1. Similarly to the above, only less than 10% of the total protein mass is bound to the column under this formulation. Furthermore, any unbound nucleic acid is washed away. After sampling, the column is washed with the same solution until the eluate A 280ra^ stops falling. The target protein and the combined proteins are then washed off in batches with the same buffer containing Imol/L of KCl. Although some tailing is still observed, running a 2-column volume buffer is usually sufficient to remove most of the protein. In most cases, this general procedure is sufficient to remove most of the nucleic acids, allowing the sample to be concentrated and the target protein to be purified by a factor of 5 to 10. The protocol can also be adapted for purification of a wider range of samples. An intermediate elution step is carried out with a solution containing a concentration of kCal sufficient to remove as much of the exogenous protein as possible, rather than the target protein. The lowest concentration, KCl, can then be used to remove the target protein, while leaving the other exogenous proteins in a bound state. This enhanced protocol requires repeated elution experiments and may fail, but usually results in 20-fold purification of the target protein. In some cases, the target protein may be so tightly bound to the analyzing medium that stronger elution conditions are required. We have found that Imol/LKBr is a good choice, but the resulting eluate should be dialyzed quickly as it may cause some protein denaturation. Alternatively, increasing the concentration of KCl to 2 m d/L may give good results. The Mi-Geel Blume column can be reused, but it should also be regenerated with 2 mOl/L guanidine hydrochloride or 1.5 mol/L sodium thiocyanate over a number of column volumes and then equilibrated with the initial buffer. Sodium azide can then be added to the column and stored at low temperature. Under these conditions, both the column and the resin can be stored steadily for several years. For more product details, please visit Aladdin Scientific website.
The ratio should be adjusted as appropriate. Higher flow rates should be used for larger diameter columns.