I am trying to purify protein having molecular weight ~50Kda under denaturing condition through Ni-NTA chromatography and got following profile. Any suggestion to stop protein coming in flow through and not binding with column.
conditions are
protein dissolved in buffer-A (tris 50mM Urea 8M, Nacl-300mM)- for loading the sample on column.
column washed with same buffer supplemented with 60mM Imidazole.
and then eluted using imidazole linear gradient from 60-800mM Imidazole in Buffer-A
Bands are-
GEL-1
Lane 1- marker
Lane 2-10 is elution during gradient
GEL-2
Lane 1- marker
Lane 2-8 is elution during gradient
Lane-9 flow through collected at the time of sample loading on column
Lane-10 -washing flow through of column by buffer A supplemented with 60mM Imidazole.
Image -3 FPLC profile
Hi,
What is the pH of your loading buffer, in which, your protein is dissoved? And what is the size of the column that you used?
Hi there,
Make sure the pH is above 6 and check the presence of Histag on your protein (WB with antiHis, sequencing of the plasmid)
What is the nominal binding capacity of your column? It could be that you are overloading the column, i.e. it can't bind that much protein.
If that's the case, you could try a larger column or less protein in your loading buffer.
Also, how many times have you used the column? Have you tried stripping and recharging the column? I am afraid, that if your column has been used for multiple of times, chances are that your column may have been clogged, which then reduces the binding capacity of your protein. That maybe the reason why you are seeing major fraction of your protein is in the flow-through. Try washing your column with 1M NaOH or try stripping and recharging the column and see if this resolves your problem?
It could be that most of the protein is severely aggregated, even in 8M urea, preventing it from binding.
You could add 20-40 mM imidazolo in the equilibrated, sample and wash buffer. Also try to use two colums in tandem and reduce wash buffer pH.
You should try to dialyse your sample first in an appropriate salt buffer with a pH around 7-8. You should dialyse for several hours with multiple buffer changes. Then prior to binding to Ni-NTA beads, add a small amount of imidazole (about 10mM). Ensure beads and sample are mixed thoroughly at 4C for at least 1 hr. And yes, as another member pointed out, ensure you are using fresh Ni-NTA beads.
You also need imidazole in your protein buffer. I usually use 5-20 mM.
If your are worried about the column, clean it with 7M GuHCl followed by 2% SDS, then lots of distilled water and finally starting buffer. My experience is that the columns get better with use, not worse, so long as you clean them once in a while and store them properly (see the manual). The whole point of these columns is that they are reusable. Your profile indicates that 60 mM imidazole is not too high. Try slowing down the loading flow rate several fold and make sure sample and loading buffers are at room temp., not cold, during the binding step.
An easy thing to try is to re-apply the flow through to fresh resin. This has worked for me a couple of times.
Also, you should check that the pH of your lysate is 8.0
Thanks to all suggestions. I would try to follow the suggestions and report the results back.
I'm currently having the same problem, what did you change in your conditions and how did it affect your results?
@ana Sofia Garcia change in pH and reducing the flow rate increased the binding on column but it also increases the amount of contaminant bands. so i moved for two step procedure for purification; i.e. Ni-NTA affinity followed by Gel filteration.
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