Hi everyone,
The DE-52 column is equilibrated with [pH 8.0 20 mM Tris-HCl, 1 mM EDTA] buffer, and the pH is around 8.5 at 4 deg (checked with pH meter). The Mb (pI ~ 7) is dialysed overnight and diluted 10 times before loading onto DE-52 column. After loading, the flow through and wash solution contains mostly of the Mb. This is checked with SDS-page and UV/Vis spectroscopy. I have also check the eluted fractions from DE-52, it only consist of a little Mb.
I am wondering why my Mb is unable to bind onto the column. Please kindly help be with this problem, thanks !
Dear Xiu-Wen,
Check the salt content of your buffer. The easiest way is to run your solution through the conductivity meter on your system. A 20 mM Tris solution should give you about 0.9 mS/cm.If it is higher, I suggest you dialyse it again.
Typically, you have to dialyse 3x at 40x the original volume. If you use a vigorous mixing, you may cut the dialysis time to 3-4 h/dialysis.
Best luck,
Wes
Activation of an anion exchanger is through equilibration. It is done by running 5CV of the binding buffer. What else do you have in mind?
Dear Wes and Jasim,
Many thanks for your advice :) ! I think I will try to deep clean DE-52 with NaOH first and to dillute my sample 40X the original volume.
Xiu-Wen
What is the protein concentration after diluting the Myoglobin loaded to the column by 10X? To begin with you stated that you dialyzed overnight, which I assume is you dialyzed versus the same Column equilibration buffer. If you dialyzed to completeness, you would not have to dilute the sample out at all, which is more preferable. Also you do need to make sure that if you are running at pH 8.0 Equilibration that the DE-52 is equilibrated to pH 8.0. Do you have your DE-52 in the H+ or OH- form? Did you Clean the DEAE first with NaOH and then follow with HCl, as this will leave it in the H+ form.
In addition, if you are only interested in purifying myoglobin, there are other methods that would not require ion exchange. Use of some traditional precipitation methods for collection and concentration, followed by simple SEC Chromatography using a SEC media that will allow the Myoglobin to emerge just after the void volume , will yield purified Myoglobin.
You may also need to consider how much total protein you have relative to the size of the column. I assume you are using a conventional column and you are using gravity flow or a peristaltic pump rather than a spin column? And that you are trying to separate the myoglobin from other proteins? On a conventional column, you will want to find the conditions such that the proteins including the myoglobin binds to the top few inches of the column. This should happen whether you start with a dilute or concentrated protein sample. Myoglobin being red or brownish-red, you would be able to see this very clearly. After you have passed enough buffer through so no unbound proteins are elating, you can elute by switching to a different buffer, either a higher concentration of the buffer, or the same buffer with added NaCl. You could set up a linear or concave gradient so that the buffer is gradually changed. Try to choose conditions that keeps the myoglobin coming down the column in a tight band.
i found an article that details conditions - size of column, protein load, buffers - used to purify some myoglobins that might be helpful http://www.jbc.org/content/236/8/2238.full.pdf
Dear Anna and Vernon,
Thanks for your answers ! I packed the column myself, and yes, I am using gravity flow. I clean the column with NaOH but I did not pull the pH back to acidic condition with HCl. Instead, I wash the column with large quantity of water and equilibrate with Tris buffer, so I think my DE-52 is in the OH- form. The concentration of Mb after I dilluted 10 times is around 36 uM. After I load the Mb onto the column, I did not see a red ring form on top of the column. Does it means that the reason my Mb did not bind is due to the fact that I did not equilibrate my column with HCl? and Mb may require H+ type DE-52 to binds?
Thank you for the article, I will read it carefully.
Xiu-Wen
Yes, that is very possible that without the H+ form, the binding is quite different. If you were to analyse in eluate from the column, after all teh Myoglobin passed through, you may have discovered some of the what I refer to as the contamination proteins.
I repeated the purification, this time I collected the flow and wash solutions. After that, they are compressed and loaded onto a SEC column. In the end I mannaged to get Mb with satisfying purity. I have also checked the elution fractions from the DE52 column with SDS page. These fractions contain mainly contamination proteins. For now, I will stick to this purification process, and I will try the H+ form later on. :)
Xiu-Wen
Good to know, all worked out. Did you achieve close to 95% purity or better?
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