Both of these two types of support are of 4% agarose matrix. Agarose 4FF is rigid, highly cross-linked polymer. It exhibits excellent chemical and physical stability and allows applications using medium pressure (up to 0.3 MPa). Therefore, the recommended flow rate is 300 - 500 cm/hour. Glutathione Agarose is non-cross-linked medium. It offers excellent binding capacity for targeted proteins. It is suitable for applications using gravity flow (with flow rate of 100 - 300 cm/hour).
Efficient, single-step chromatography purification of Maltose binding protein (MBP) tagged fusion proteins with high yield and purity. Stable and resistant to amylase degradation. ... Read more
PROTEINDEX™ Dextrin Agarose 6FF is a chromatography medium for purifying proteins fused to maltose binding protein (MBP-tagged protein). Recombinant proteins with MBP-tags often gives increased expression levels and higher solubility of the target protein. Proper folding of the attached protein has also been shown to be promoted by the MBP tag. Since MBP increase the solubility, the tag is extremely useful for recombinant proteins accumulated in an insoluble form (inclusion bodies).
Dextrin is the polymeric product resulted by enzymatic hydrolysis of amylose with beta amylase. Compared to Amylose that is susceptible to amylase degradation, Dextrin is very stable. When used as the ligand substrate immobilized on the resin for purification of the MBP tag, Dextrin offers consistently strong affinity absorption even after repeated uses.
The base matrix of Dextrin Agarose 6 Fast Flow is a robust, highly cross- linked 6% agarose. The crosslinking of the base matrix has been optimized to give high physical and chemical stability, high specificity of the binding.
Affinity purification using Dextrin Agarose 6 Fast Flow take place under physiological conditions and mild elution is performed using maltose which preserve target protein activity. These mild conditions may even allow purification of intact protein complexes.
Our proprietary ProteIndex™ coupling technology ensures stable immobilization of active ligand and minimal ligand leakage in a wide range of purification buffer conditions. The resins can be reused multiple times to purify the same target protein.
- High selectivity for high purity
- > 10 mg MBP tagged protein (80 kDa)/mL medium
- Stable coupling of ligand, low leakage; can be re-used to purify the same protein
Figure 1. Purification performance of Dextrin 6FF resins.
50 mL of E.coli lysate containing a MBP-tagged fusion protein (~ 80 kDa) was loaded on 0.5 mL packed Dextrin 6FF resins at 0.5 mL/mL. The column was then washed with 20 mL of Binding Buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH7.4). The target MBP-tagged protein was eluted with 9.5 mL of elution buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 10 mM maltose, pH7.4). Fractions of lysate, flow-through, and eluate were analysed by SDS-PAGE. Lanes: (1) Raw cell lysate; (2) Flow-through; (3) Eluate.
|Binding capacity||>10 mg MBP tagged protein (80 kDa)/mL medium|
|Matrix||6% highly cross-linked agarose supplied as 50% slurry|
|Particle size||45 μm - 165 μm|
||0.3 MPa (3 bar, 43 psi)|
|Recommended flow rate
||300 - 500 cm/hour|
||2 - 8°C|
||20% ethanol as preservative|
Product is shipped at ambient temperature. Upon receipt store at 2 - 8°C. Do not Freeze.
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Q&As and Reviews
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Customers Questions & Answers
Yes. Glutathione Agarose has high binding capacity for GST-tagged fusion proteins or other glutathione binding protein. The medium can be directly added to pre-treated cell lysates to capture target proteins.
There are many factors that can affect the final purification product. First of all, the composition of the buffers being used in binding, washing, and elution may affect greatly the yield and the purity of the final product. These buffers to should be compatible with the medium. Chemicals, such as 1% Triton X-100, 1% Tween-20, 1% CTAB, 0.03% SDS, or 0.1% NP-40, will not significantly affect the binding of GST or GST-fusion protein to Glutathione Beads. Therefore, these chemicals may be used to reduce non-specific binding. Adding DTT to a final concentration of 1 - 10 mM may greatly increase binding of certain GST-tagged proteins to Glutathione Agarose medium. However, while 1 - 10 mM DTT can be included in the binding and elution buffer to increase purity, it may result in lower yield of some target proteins. Binding of GST-tagged proteins to the chromatography medium depends on its size, conformation, as well as its concentration in the sample loaded. Binding of GST to glutathione is also flow dependent. Lower flow rates often increase the binding capacity, whereas higher flow rates decrease the binding efficiency. Therefore, lower flow rates are recommended, in particular, during sample loading. Furthermore, protein characteristics, pH and temperature may also have impact on the binding capacity.
The answer is no if the target protein is GST-tagged fusion protein and it requires functional GST to bind to the glutathione ligand. The reason is that the GST tag will be denatured in 8 M urea (the same case is for guanidine-HCl) and, even the denaturant is removed, will not be refolded into the functional conformation to bind to the glutathione ligand. Therefore, neither 8 M urea nor guanidine-HCl is recommended in any solutions in the purification procedure. If the presence of denaturant is indispensable for certain applications, urea can be added to a final concentration of up to 1 M, however, at a cost of lower yield of the final product.
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