Question
Asked 2nd Jul, 2013

Protein is getting eluted in Flow through

My gene of interest cloned in pET28a with C-terminal His tag is expressed in arctic express E.coli strain. Protein is in soluble form and using GE fast flow Ni-NTA beads for purification. My protein of interest is getting eluted in flow through. Please help me in this regard

Most recent answer

13th May, 2020
Robert Adamu Shey
University of Buea
May you need to review the composition of your lysis buffers the presence of imidazole could be hindering protein-Ni2+ interactions. On the other hand, the column may just be saturated so you may need to reduce the amount of lysate that you are passing through the column
2 Recommendations

All Answers (11)

2nd Jul, 2013
Alejandro Gomez Mejia
University Hospital Zürich
Hello Chandra, I would like to know if you have worked with this protein before, maybe the retention time and the purification protocol is not well standarized, also I think you should first check your column in the case is not new, maybe the packing is not well reactivated or has some imidazole in it, else maybe you should try using different concentrations of imidazole.
Also did you check on an SDS-gel to see if the protein weight is corresponding to your protein plus the his tag?, just to check.
I have worked with protein purification before and I had this problem and solved by gradually increasing the imidazole concentration.
Good luck
1 Recommendation
3rd Jul, 2013
Chandrasekaran Sambamurthy
The University of Arizona
No, this is the first time I am working with this protein. The beads are new and I have used wide range of imidazole concentrations till 500uM. I have done both SDS and western blot to confirm my protein before loading onto the column.
Does imidazole in the binding buffer affect the binding of protein to the column?
3rd Jul, 2013
Daniel M Cohen
Spark Therapeutics, Inc.
Yes, often you need to lower the amount of imidazole in the binding buffer. How much imidazole are you using for your binding buffer?
1 Recommendation
3rd Jul, 2013
Chandrasekaran Sambamurthy
The University of Arizona
I am using only 10mM imidazole in binding buffer.
Does some amount of protein will be lost in the flow through during the purification process?
1 Recommendation
3rd Jul, 2013
Daniel M Cohen
Spark Therapeutics, Inc.
Do you have ionic detergents, E. coli genomic DNA, or metal chelators such as EDTA in your lysates? Any of these components can inhibit protein binding to NTA columns. If these are not present, the problem may be one of limited accessibility of your C-terminal tag to the HisBind resin due to protein conformation (most often, oligomerization of your recombinant protein). To improve tag accessibility, you can add urea or GuHCl as chaotropes to partly or complete denature your protein. If denaturants improve binding, then you can be sure tag accessibility is the problem.
1 Recommendation
3rd Jul, 2013
Alice Dawson
University of Dundee
What pH are you using? Optimal binding is usually pH 7.5 - 8 ish.
Are there any structures known of closely related proteins so you can check the location of the C-terminus? I know I have realised too late in the past that the tag was not accesible!
3rd Jul, 2013
Alejandro Gomez Mejia
University Hospital Zürich
What Daniel says is true, you should also check the components remaining in the lysate as they might be interfering with the binding, using Urea might be a good idea but only if you dont need your protein for any further activity assays a this will denature the protein and I dont know if its possible but i think is too hard to control how the protein will refold afterwards.
My best guess is that the C-terminus of your protein is not so accesible, so one idea would be to control the flow and use a slow flow to allow a higher retention time of your protein and increase the chances of the C-terminus to bind to the NTA column, the imidazole concentration in the binding buffer is not so high so there should be no problems there, I usually use 20 mM for the binding buffer and 500 mM for the elution buffer.
Good luck
1 Recommendation
4th Jul, 2013
Vineet Kumar
Institute of Microbial Technology
Hi Chandra,
I don't know what kind of protein you are expressing. Like I faced the same problem with one protein. To overcome that problem I induced at lower O.D. like0.3.
If your protein is of general use of cell e.g. some polymerase , in that case chromosomal segment of gene will also express your protein and you will get non tagged protein . If you will give induction at higher O.D. then your non tagged protein will out compete with recombinant one and will not bind to column because of lack of availability of tag. Induction at lower O.D. will enhance the more population of your recombinant protein..It may be one of the solution to your problem...thanks
4th Sep, 2019
Kamalika Samanta
The University of Tennessee Health Science Center
I am trying to purify his-tag protein but my protein is in supernatant, after purification, I find higher concentration in the flow through. Please suggest to troubleshoot this
1 Recommendation
12th May, 2020
Hafeez Wazir
Gomal University
I am trying to purify GST-Target protein , the flow through drops are very slow. Please suggest to troubleshoot this

Similar questions and discussions

Why is my protein both in the flow-through and unable to be eluted?
Question
8 answers
  • Jerry WangJerry Wang
During ni-nta purification of my his-tagged protein, I collected sample from each step and ran them on a sds-page. I see my protein band in the flow-through (thickness indicate about 30% of total protein content), as well as in the first wash. This band then becomes progressively lighter with subsequent wash. When I got to the elution step, I did not get any protein. Finally, I added sample buffer directly to the beads and ran run on the gel as well and saw my protein band (around 30% of total protein content). 
Here are more details:
I used 100 microliter suspension of newly-charged nickel beads (50%) for a 5ml culture (= 1ml lysate).
All buffers were freshly prepared:
Binding buffer:
  • 20 mM Tris-HCl pH 8.0
  • 0.5 M NaCl
  • 20 mM imidazole
Washing buffer: everything remains the same except 40mM imidazole
Elution buffer: everything remains the same except 250mM imidazole
I incubated the protein supernatant with the beads for 1.5hr before centrifuging at 3500 rpm for 3mins and aspirating the supernatant
I also incubated the beads with the elution buffer for 10 minutes before each elution step.
Here is the gel image: I apologize for the poor quality. The 1st column on the left is the flow through and my band is the one at the red marker. You can see the band in the next lane, which is the first wash, followed by 2nd wash, 1st elution, 2nd elution, and the uneluted sample. The next lane with lots of protein is the flowthrough of a second protein which is very similar to the first one, you can see that once again most protein is lost in the first wash, but some still remains on the beads after elution.
Thank you for your help, this problem has been bugging me all day.

Related Publications

Got a technical question?
Get high-quality answers from experts.