I am doing cation exchange chromatography by using HiTrap-SP-HP column, and the buffer A is 20 mM Tris, 50 mM NaCl, 1 mM MgSO4, 1 mm DTT. Since the FPLC machine is old, after loading the protein, I am doing the fractionation by manual step by step increase of Buffer B (Buffer A + 0.5 M NaCl) percentage using one pump and the absorption of 215 and 280 is estimated as well as Dot-blot for all fractions. Sometimes it works, however, sometimes target protein elute in all fractions (about 200 ml) instead of a nice peak (20 ml). Could it be due to the unclean column? However, we cleaned the column rigorously with EtOH and NaOH. I am wondering what else can be the reason. Is it possible in case of a very slow increase of Buffer B percentage, the protein of interest elute very slowly without showing a traceable peak?
Thank you in advance for sharing your experience.
Problem is interesting however you have taken all possible measures. Please refer :
pohl C. 2013.Recent development in ion exchange columns for ion chromatography.LC-GC 31 ,
This elution profile can arise if the protein is highly aggregated. You can check for this by doing gel filtration chromatography.
Dear Adam B Shapiro
Thank you very much. This is actually a very likely possibility since my protein of interest is an amyloid aggregation-prone protein. Is there any source that describes the mechanism and the way to avoid? Do you think this aggregation occurs during the loading of protein into the column or during the elution when local concentration arise intensively?
The aggregation probably occurred during expression of the protein. If the protein is already known to aggregate, this may not be preventable by changing expression conditions, such as lowering the temperature. You may be able to purify it as a monomer in 8M urea, which does not interfere with ion exchange chromatography (as long as the urea solution is fresh), but it will probably aggregate again when the urea is removed.
Dear Adam B Shapiro
Thank you for your response. Since this protein is aggregation-prone we are always careful about aggregation, and the protein extract we work on is always filtered (0.2 um) and is completely transparent, especially before chromatography. Is it possible that some small soluble aggregates (oligomeric forms) interfere with chromatography?
0.2 µm is 200 nm, which is still large enough to pass substantial aggregates. A small protein molecule (~30 kDa) has a hydrodynamic radius of only 2-3 nm.
Dear Amir
You have mentioned that buffer A contained 50 mM NaCl is this correct? if so then that's a problem you need NaCl in buffere B only but not in buffer A in order to compete the target protein which is stick to ion exchanger but that doesn't happened in your case because of the presence of NaCl in buffer A and that's why target protein eluted in all fractions. so ommit NaCl from buffer A and give it another try, good luck
Hope this help
Dear Abdulhadi Albaser
Thank you for your response. In the standard protocol for this protein Buffer A contains 50 mM NaCl, and then elution is with an increasing percentage of Buffer B with 500 mM NaCl. Also, the protein of interest is not in the flow-through fraction meaning that it binds entirely to the column. Thus, 50 mM NaCl is not preventing the binding of target protein but some other contaminating protein with lower affinity to cation exchange column. And as I mentioned, the problem is not consistent, sometimes the process is completely fine, and I have a nice peak of the target protein, but sometimes this problem happens. So the general protocol is not problematic.
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