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Rose BioPak® MicroColumns™ Filled with DEAE Resin
BioPak DEAE filled MicroColumns utilize ion-exchange chromatography principles to isolate and purify biomolecules based on their charge. Applications include isolating proteins, purifying PCR products from amplification reactions, cleaning-up DNA from enzymatic reactions, and purifying plasmid DNA from bacterial lysates. These purification applications are typically performed prior to protein digestion, cloning direct sequencing reaction, DNA amplifications, gel electrophoresis, HPLC, mass spectrometry and other techniques.
General Principles
The target molecule is bound to the DEAE resin in a low salt and/or low pH buffer solution, then eluted using a high salt and/or high pH buffer solution. This process depends upon the interaction between the charge of the target molecule and the opposite charge of the DEAE resin. Varying the ionic strength and/or pH of the buffer solution controls this interaction. In a low salt and/or low pH buffer solution, competition for charged groups on the DEAE resin will be at a minimum, and the target molecule is bound strongly. As the ionic strength and/or pH of the buffer solution increases, competition for charged groups on the DEAE resin also increases. Simultaneously, this reduces the interaction between the DEAE resin and the target molecule, resulting in its elution.
Advantages
DEAE-filled BioPak MicroColumns are a rapid and inexpensive technique for isolating and purifying a wide range of biomolecules from various sources. Isolation is accomplished using one of two strategies. Either, unwanted molecules in the sample are adsorbed to the resin, and the molecule of interest easily passes through the
MicroColumn. Or, the molecule of interest is bound to the resin, in a low salt and/or low pH buffer solution, and then is simply eluted using a high salt and/or low pH buffer solution, and then is simply eluted using a high salt and/or high pH buffer solution. Both of these strategies ensure excellent recovery of the target molecule.
The high speed of the DEAE-filled MicroColumn purification process increases the potential to recover biologically active molecules. BioPak MicroColumns are easily adapted for various applications using a 200ul hand-held pipettor. And, because they are mounted from a rack onto a pipettor, MicroColumns never need to be handled directly, eliminating enzyme contamination.
Technical Information
Matrix: Microgranular Cellulose
Particle Size: 60-70um
Pore Size: N/A
Binding Efficiency: 90-95%
Elution Efficiency: 85-90% (using optimal pH binding and elution solutions, per detailed protocols)
Protocol Buffers
Preparing: DEAE-filled BioPak MicroColumns are prepared by cleaning and hydrating the resin using various salt/buffer solutions. When cleaning the resin, use a high salt buffer to remove any unwanted substances. Residual NaCl can then be washed from the MicroColumn with a non-salt buffer. A low salt buffer is used to present ideal conditions for capturing the target molecule. To maintain hydration of resin, never completely dispense salt/buffer solution from the MicroColumn.
Equlibrating and Binding: Equilibrate by bringing the ionic composition of the sample to that of the binding buffer solution. This allows the target molecule (carrying the appropriate charge) to displace counter-ions and bind reversibly to the resin. When choosing a buffer, use the highest ionic strength that allows the target molecule to bind to the DEAE resin, while preventing other molecules from binding.
Washing: Unwanted molecules, that bind to the resin with less affinity than the target molecule, should be washed from the DEAE-filled BioPak MicroColumn. This can be done by using a buffer solution unfavorable to the binding of unwanted molecules, while still favorable to the binding of the target molecule. To accomplish this, increase the ionic strength and/or change the pH of the wash buffer solution beyond the ionic strength and/or pH of the binding buffer solution.
Eluting: The target molecule is eluted from the DEAE-filled BioPak MicroColumn in a buffer solution unfavorable for ionic bonding of the target molecule. This involves increasing the ionic strength and/or the pH of the elution buffer solution beyond that of the wash buffer soultion. When choosing a buffer, use the lowest ionic strength that causes the target molecule to elute, while leaving the maxium number of unwanted molecules bound to the resin.
The working pH range for the DEAE-filled BioPak MicroColumn is between 2 and 9. Ideal pH for these MicroColumns is between 4 and 9. These buffers often need purification before use, and are sensitive to temperature change. Commonly used buffers are N-methyl piperazine (pH 4.5-5.0), bis-Tris (pH 5.0-6.4), Tris-EDTA (TE) (pH 7.4-7.8), and diethanolamine (pH 8.4-8.8).
Protocol Guidelines
In summary, preparation for cloning quality templates of dsDNA simply requires the following steps. First, prepare the DEAE resin in the MicroColumn by pipetting up and down with several different salt/buffer solutions. Equilibrate the sample by bringing the ionic composition to that of the appropriate binding/loading buffer solution. next, bind to the DEAE resin by pipetting the sample up and down several times with the same binding/loading buffer solution. Finally, elute the sample from the DEAE resin by pipetting up and down several times with a high salt buffer solution.
We have found that multiple elutions of smaller volumes will ensure greater recovery than a single elution of a larger volume. And, if the quality of the sample in the loading solution is greater than the capacity of the DEAE-filled MicroColumn, the process of preparing, equlibrating, binding, washing, and eluting may be repeated as many times as necessary with the same BioPak MicroColumn.
Rose DEAE Resin Filled MicroColumn Protocol
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