For Anja’s question, I think the difficult thing in CE-SELEX is to determine at which point we should cut the collection. If using a Flu-mag SELEX, for example, after incubation of ssDNA library with magnetic beads, all we have to do is to wash away the unbound by elution. However, in CE before the major peak of ssDNA library one can not observe peak of bound DNA with extremely low Kd (concentration is too low). One can also collect DNA bound with low Kd or other forms of DNA even not bound with protein before the major peak.
The Kd was determined using an ultrafiltration assay and radioactively labeled aptamers. Is there a safer method? Could other techniques like affinity CE used?
To determine Kd with a safer method, I am wondering could we use fluorescence polarimeter? Because polarizations of pure protein and ssDNA bound protein should be different,we can determine the concentration by different polarization properties as we discussed in unit 1&2.
The reason we choose ultrafiltration instead of affinity CE is that aptamer and protein has a strong bio-interacton. The Kd is uauslly about nM level. This low Kd determined that we need to study the interactoin when the concentration of the ligand and protein are at nM level. Capillary Electrophoresis with a laser induced fluorescence usually has a detection limit of 0.1 nM. If we use a nM level protein or ligand, when it is injected to the capillary, it is diluted. So it may just reach the detection limit or even not been seen. So we need to use other method with a low detection limit to get an accurate Kd.
The reason we choose ultrafiltration instead of affinity CE is that aptamer and protein has a strong bio-interacton. The Kd is uauslly about nM level. This low Kd determined that we need to study the interactoin when the concentration of the ligand and protein are at nM level. Capillary Electrophoresis with a laser induced fluorescence usually has a detection limit of 0.1 nM. If we use a nM level protein or ligand, when it is injected to the capillary, it is diluted. So it may just reach the detection limit or even not been seen. So we need to use other method with a low detection limit to get an accurate Kd.
The reason we choose ultrafiltration instead of affinity CE is that aptamer and protein has a strong bio-interacton. The Kd is uauslly about nM level. This low Kd determined that we need to study the interactoin when the concentration of the ligand and protein are at nM level. Capillary Electrophoresis with a laser induced fluorescence usually has a detection limit of 0.1 nM. If we use a nM level protein or ligand, when it is injected to the capillary, it is diluted. So it may just reach the detection limit or even not been seen. So we need to use other method with a low detection limit to get an accurate Kd.
Assorted comments that may have already been discussed in class:
1. One of the great advantages of CE-SELEX is that the target molecule is not sterically hindered due to immobilization of the target. This advantage makes it appealing even when it is difficult to select a point in which bound and unbound aptamers elute. At any rate, having several rounds of SELEX addresses this issue.
2. Fluorescence polarization would be a good alternative to radioactively labeling when the size of the aptamer and the target are significantly different.
3. I agree that the LOD of the instrument is an important factor. The LOD in CE-LIF may compromise our ability to use it in determining low Kd values. Why would the sample need to be diluted though?
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Comments
I am just wondering what the limitations of using this method would be as opposed to methods that are currently used? Thanks.
Posted by: Anja Lesaja | April 20, 2008 02:03 PM
For Anja’s question, I think the difficult thing in CE-SELEX is to determine at which point we should cut the collection. If using a Flu-mag SELEX, for example, after incubation of ssDNA library with magnetic beads, all we have to do is to wash away the unbound by elution. However, in CE before the major peak of ssDNA library one can not observe peak of bound DNA with extremely low Kd (concentration is too low). One can also collect DNA bound with low Kd or other forms of DNA even not bound with protein before the major peak.
Posted by: Meng Jing | April 22, 2008 09:46 AM
The Kd was determined using an ultrafiltration assay and radioactively labeled aptamers. Is there a safer method? Could other techniques like affinity CE used?
Posted by: Edgar Arriaga | April 22, 2008 10:51 PM
To determine Kd with a safer method, I am wondering could we use fluorescence polarimeter? Because polarizations of pure protein and ssDNA bound protein should be different,we can determine the concentration by different polarization properties as we discussed in unit 1&2.
Posted by: Meng Jing | April 23, 2008 12:41 PM
The reason we choose ultrafiltration instead of affinity CE is that aptamer and protein has a strong bio-interacton. The Kd is uauslly about nM level. This low Kd determined that we need to study the interactoin when the concentration of the ligand and protein are at nM level. Capillary Electrophoresis with a laser induced fluorescence usually has a detection limit of 0.1 nM. If we use a nM level protein or ligand, when it is injected to the capillary, it is diluted. So it may just reach the detection limit or even not been seen. So we need to use other method with a low detection limit to get an accurate Kd.
Posted by: Jing Zhang | April 23, 2008 10:49 PM
The reason we choose ultrafiltration instead of affinity CE is that aptamer and protein has a strong bio-interacton. The Kd is uauslly about nM level. This low Kd determined that we need to study the interactoin when the concentration of the ligand and protein are at nM level. Capillary Electrophoresis with a laser induced fluorescence usually has a detection limit of 0.1 nM. If we use a nM level protein or ligand, when it is injected to the capillary, it is diluted. So it may just reach the detection limit or even not been seen. So we need to use other method with a low detection limit to get an accurate Kd.
Posted by: Jing Zhang | April 23, 2008 10:49 PM
The reason we choose ultrafiltration instead of affinity CE is that aptamer and protein has a strong bio-interacton. The Kd is uauslly about nM level. This low Kd determined that we need to study the interactoin when the concentration of the ligand and protein are at nM level. Capillary Electrophoresis with a laser induced fluorescence usually has a detection limit of 0.1 nM. If we use a nM level protein or ligand, when it is injected to the capillary, it is diluted. So it may just reach the detection limit or even not been seen. So we need to use other method with a low detection limit to get an accurate Kd.
Posted by: Jing Zhang | April 23, 2008 10:49 PM
Assorted comments that may have already been discussed in class:
1. One of the great advantages of CE-SELEX is that the target molecule is not sterically hindered due to immobilization of the target. This advantage makes it appealing even when it is difficult to select a point in which bound and unbound aptamers elute. At any rate, having several rounds of SELEX addresses this issue.
2. Fluorescence polarization would be a good alternative to radioactively labeling when the size of the aptamer and the target are significantly different.
3. I agree that the LOD of the instrument is an important factor. The LOD in CE-LIF may compromise our ability to use it in determining low Kd values. Why would the sample need to be diluted though?
Posted by: Edgar Arriaga | April 26, 2008 09:20 AM