New England Biolabs, E8022S, Amylose Resin High Flow

Amylose Resin High Flow is a cross-linked affinity matrix used for the isolation of proteins fused to maltose-binding protein (MBP). This rigid matrix can be used in automated chromatography systems. Related Categories Amylose Purification (MBP-tag),, Affinity Purification,, Protein Purification Applications MBP Affinity Tag,, Affinity Purification & Expression Tags,, Protein Purification, Specification Storage Buffer 20% Ethanol Binding Capacity >4 mg MBP5* -paramyosin δSal fusion protein / ml amylose high flow resin. Can be Regenerated Yes FAQ Q: What gravity column do you recommend? A: We recommend the 2.5 cm (internal diameter) Econo Column line from Bio-Rad or the Kontes Flex-Column. Column sizes (internal diameter and length) can be scaled up or down and should be based on the amount of resin needed to isolate your protein of interest in sufficient yields. Q: Much of my fusion protein flows through the amylose column. Is there anything I can do to improve my fusion’s affinity for the amylose column? A: A MBP fusion protein might not stick to the amylose column because of the presence of some factor in the extract that interferes with binding, or because of a low intrinsic affinity. Factors in the crude extract that can interfere with binding include nonionic detergents and cellular components that are released during alternative methods of lysis (prolonged treatment with lysozyme or multiple passes through a French press). In addition, cells grown in LB and similar media have substantial amounts of an amylase that interferes with binding, presumably by either cutting the fusion off the column or by releasing maltose that elutes the fusion from the column. By including glucose in the media, expression of this amylase is repressed and the problem is alleviated. A low intrinsic affinity could be caused by an interaction between the protein of interest and MBP that either blocks or distorts the maltose-binding site. Although this may be inherent in the protein of interest, sometimes the problem can be alleviated by shortening or lengthening the polypeptide that is fused to MBP. Q: How many times can I use the amylose column? A: The most important variable in determining the useful life of the amylose resin is the amount of time it is in contact with trace amounts of amylase present in the crude extract. Under normal conditions (crude extract from 1 liter of cells grown in LB + 0.2% glucose, 15 ml column), the column loses 1–3% of its initial binding capacity each time it is used. If the yield of fusion protein under these conditions is 40 mg, this means that after 3 to 5 runs there would be a decrease in the yield. In practice, we often use a column 8 or 10 times before we notice a significant drop in the yield. Q: What is known about binding in the presence of nonionic detergents? A: Some fusion proteins do not bind efficiently (< 5% binding) in the presence of 0.2% Triton X–100 or 0.25% Tween 20, while other fusions are unaffected. For one fusion that does not bind in 0.25% Tween 20, diluting the Tween to 0.05% restores about 80% of the binding. Q: Can I substitute a different buffer and/or salt concentration in the Column Buffer? A: Yes, HEPES, MOPS, and phosphate buffers (at pH values ranging from 6.5 to 8.5) can be used instead of Tris-HCl in the Column Buffer with similar results. NaCl or KCl concentrations of 25 mM to 1 M are also compatible with the affinity purification. Q: I see my intact fusion protein by SDS-PAGE when I run cells boiled in Sample Buffer, but when I check the crude extract the fusion is degraded. A: For fusions expressed in the cytoplasm, in many cases most of the degradation happens during harvest and lysis. Harvesting promptly and lysing the cells quickly may help. In other cases, degradation occurs when the fusion protein is exposed to periplasmic or outer membrane proteases. The best strategy in either case is to use a host which is deficient in the offending protease(s). Q: When I run my purified fusion protein on SDS-PAGE, why do I see multiple bands instead of a single band of the expected MW? A: There are two likely explanations for this result. The first is that the fusion protein is unstable, which most often leads to degradation in vivo. In this case, one would expect to see bands between the size of MBP (42.5 kDa) and the size expected for the full-length fusion, since fragments smaller than MBP would not bind to the affinity column. An exception would be if the fusion protein breaks down at the junction between MBP and the protein of interest, and the protein of interest oligomerizes. In this situation, the protein of interest may bind to the fusion protein, and therefore a band the size of the protein of interest can appear even if it is smaller than MBP. The second explanation is that the protein of interest is binding non-specifically to other E.coli proteins, e.g. it has a surface that binds other proteins by electrostatic or hydrophobic interactions. In this case, modifications to the column buffer can sometimes be used to help wash the interacting proteins away. Electrostatic interactions can be weakened by including up to 1 M NaCl in the column buffer, and hydrophobic interactions can be weakened by lowering the salt to 25-50 mM NaCl and including 5% ethanol or acetonitrile in the column buffer. Non-ionic detergents can also be used to weaken hydrophobic interactions, but they can interfere with the affinity of certain fusion proteins. Q: Can I perform a batch purification using the amylose resin? A: Yes, batch purification works well, although it is difficult to wash all the nonspecific proteins away as effectively as in a column due to the included volume in the resin. The resin can withstand centrifugation at up to 6000 x g. A good compromise is to load the resin in a batch mode, by incubating with shaking for 2 hours to overnight, then pour it in a column to wash and elute. Dilution of the crude extract may not be critical for loading the column by the batch method. Q: Is the amylose resin damaged by storage at -20°C? A: We do recommend the beads be stored at 4°C and not ever frozen The resin will freeze at -20°C but the performance of the resin is not degraded by one freeze/thaw cycle. Freezing the product in a solution other than our storage solution (i.e. in the absence of 20% ethanol) can result in reduced performance/degradation of the resin, so functional performance should be verified by the customer prior to use. Q: What is the recommended flow rate and maximum pressure for Amylose Resin High Flow once packed into a column? A: Amylose Resin High Flow has the following recommended flow rates and pressure parameters: 2.5 to 5 mL / min at < 0.1 MPa using buffers with the same viscosity as water. Q: Can MBP fusions be purified in the presence of denaturants like urea or guanidine-HCl? A: No, MBP’s affinity for amylose and maltose depends on hydrogen bonds that in turn are positioned by the three-dimensional structure of the protein. Agents that interfere with hydrogen bonds or the structure of the protein interfere with binding as well.