Hi,
I am doing an affinity chromatography protein purification for a protein of interest. I made the error of leaving my pump on too long and my buffer solution ran dry and made my column run dry to the point where the top layer of my resin bed looked dried up, but the some of the bottom half was still hydrated.
Have compromised my protein? What exactly happens when the resin runs dry?
When the resin runs dry, air gets into the column. This prevents your protein from reaching the resin, or can prevent you from eluting the protein off of the resin. Ni-NTA is an affinity chromatography, so you can still save stuff- this next step wouldn't work with gel filtration and such. Stir your entire column into solution, and let settle. That should remove the air bubbles and you can move on with life. Now, as for any dried out protein, that depends on the protein.... ;)
When the resin runs dry, air gets into the column. This prevents your protein from reaching the resin, or can prevent you from eluting the protein off of the resin. Ni-NTA is an affinity chromatography, so you can still save stuff- this next step wouldn't work with gel filtration and such. Stir your entire column into solution, and let settle. That should remove the air bubbles and you can move on with life. Now, as for any dried out protein, that depends on the protein.... ;)
I agree with Jennifer Gibbons answer. Otherwise in my opinion during operation step, you can suck the pump first to remove the air and continue run with binding buffer while hold the step until the resin ok. If top layer dried, try gave downflow. Based on my experience it helpful.
Hi Barney.
With high-load affinity columns like Ni-NTA there is a very good chance that your protein has bound (if it went dry on loading). What you do next will depend on what type of column you have. If it is a cartridge, you do not have so many options. You can try reverse flowing with loading buffer. If this does not work and you can access the top of the column, add some loading buffer and very gently stir the gel into a slurry until you get rid of the cracks. Let the resin settle and flow some more loading buffer. When you are satisfied that the column looks uniform again (a flashlight can help), you can continue with your protocol and elute. Pumping a column dry happens and does not have to mean a lost sample. With BioRad or Pharmacia-type columns you can even completely remove the resin (with protein bound) and repack as long as you keep loading buffer in contact with the protein-resin.
Good Luck and let us know how it turns out.
I would like to suggest the use of other more harder gels.
Oizumi J, Hayakawa K. Purification of the membrane protein enzyme lipoamidase by affinity chromatography. Biomedical Chromatogr 3: 274-275, 1989.
Although relatively large as compared to the HPLC silica gel (5-10 μm gel-particle size), Cellufine Chelate (iminodiacetic acid-bound cellulose gel, c.a. 120-210 μm gel-particle size) may be of choice. Although particle size of Ni-NTA Superflow Agarose (60-150 μm gel-particle size) is similar to Cellufine Chelate , the cellulose-based gel (Cellufine ) may be harder than agarose gels. Please, see the following informations; i.e.,
JNC CORPORATION、Tokyo, Japan
http://www.jnc-corp.co.jp/fine/en/cellufine/index.html
http://www.jnc-corp.co.jp/fine/en/cellufine/grade/grade-1-chelate.html
Hi, if bottom layer is still hydrated, invert the column and keep the hydrated layer at top and pass your buffer for an hour, that will clear the airlock and protein can be eluted and reused.
In general, I would recommend gravity flow for Ni-NTA columns, combined with a step gradient. Then you have little risk of running the material dry...also, many people use far to much Ni-NTA in their purifications, leading to impurities binding. If you minimise the ml of Ni-NTA gel slurry, you usually get much better purification, and you can just throw the Ni-NTA away if something goes wrong.
After reading the suggestions received till now, I would recommend to reverse the flow rate, apply a very slow flow rate only with binding buffer, and try to get rid off the air. If it did not work, you can do what Kurt has suggested. However, I also would like to emphasize to forget the peristaltic pump and to use only gravity flow in Ni-NTA affinity chromatography.
A good method for regeneration of a dry column or a column with air bubbles, which works fine in our labs, is putting the column without stop plugs in a beaker, and filling the beaker with storage buffer, so that both ends of the column are in the buffer. Then put the beaker in an exsiccator and apply vacuum, e.g with a water-jet vacuum pump. The air from the column will be removed directly.
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