Without knowing that your protein is expressed, either in inclusion bodies or in the cell membrane, it might be a waste of time trying to optimize the elution conditions. Since your protein is HIS-tagged you can use an anti-HIS antibody for for Western blot detection. Once you know whether and where your protein is expressed, you can check also by Western blotting if your protein binds to Ni-NTA or if your tag is not accessible.

Hi there,

If you are sure your protein is properly expressed, extracted and adsorbed on NiNTA column, you can have a go running a gradient of imidazole onto your NiNTA column to try to resolve the mixture of proteins.

Thankyou. I am getting good expressions of this protein, but I am stuck in purification by using Ni-NTA.

If your protein is well expressed, there are two likely possibilities:

a) it does not bind well to Ni-NTA, then you should find most of your protein in the flow through after the binding step

b) your protein binds strongly to Ni-NTA, if you are using Ni-NTA agarose beads, you can treat them with hot SDS and EDTA (Laemmli buffer) and run it on a Western blot

In the first case, you should move or change your tag, in the 2nd case you could try eluting with EDTA.

Okay, Thankyou Sir. 

There is a third possiblity:

I assume you check your protein with some kind of activity assay (as no mention of 6xHIS use for Western was made). Purification of hydrophobic proteins can be problematic with this regard, as you need to consider its solubilization from membranes (in regards of detergent type; detergent % [v/v], and detergent:protein ratio). This may CRITICALLY impair the protein's activity, esp. without reconstitution. Do have a peak into my PhD thesis: https://www.researchgate.net/publication/27559965_Characterization_of_the_Cardiolipin_Synthase_from_Arabidopsis_thaliana

Article Characterization of the Cardiolipin Synthase from Arabidopsis thaliana

try to use some other tags.. (MBP or SUMOtag), and also high salt,  these might improve solubility, apart from other suggestions. membrane proteins are very difficult to purify hence you have to play with salt, pH and tags. best of luck

Dear Priyanka,

It is not excluded your protein is migrating on the mass of 47, the bands below are its degradants. Simple comparison of total bacteria protein extracts induced and befor induction can give you a clue.

Regards - jan

Thankyou for all the suggestions. 

Hello Jan, proteins visible below my protein could be its degradants but few proteins are coming above that also. 

Dear Priyanka,

As you have mentioned that your protein is an integral membrane protein, there is almost full chance that it is going to the inclusion bodies.

You can have an idea of checking its localization in bacteria fairly simply by trying to look for expressed protein bands in SDS PAGE for supernatant and pellet fractions of cell lysate.

For membrane proteins and inclusion body proteins, full or partial denatured purification is often the only way. Qiagen has a good guide on their Ni-NTA protocol book about denatured purification. You can look for such guidelines from other companies like GE or Sigma who supplies materials for protein purification.

Also please ensure your lysis buffer has either of UREA (8M max) or Guanidium hydro-chloride (6M max). Start with a 2-3M concentration and then gradually increase by 0.5M and check for yield until you reach your desired level of purification. Adding small amount of Lysozyme, CHAPS and Triton X100 also helps. If you have to take this way of denatured purification, you have to follow gradual dialysis method to gradually lower the detergent level. Don't go for 1 step dialysis like 10ml of protein in 2L of buffer as it would abruptly lower the detergent level causing your proteins to aggregate and precipitate. Rather to remove the detergent, go for dialysis with buffer volume 5-10 times of your protein volume and change the buffer ever 0.5hr (for first 3-5 times) to 1-2hr (next 5-10 times) and then finally keep it in dialysis overnight in a buffer with 500ml-1L volume. The reason for this is to allow you protein sufficient time to fold in a cell-free system as the detergents, which actually keeps it in solution during purification, goes away.

You can check the following links:

http://proj1.sinica.edu.tw/~tigpcbmb/course%20material/cb931130/cb30.pdf

http://www.biocompare.com/4-Protein-Forums/724-membrane-protein-purification-problems/

http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1336168762999/litdoc28909531_20140915105329.pdf

Wish you best of luck,

An important issue not yet addressed, is the location of the HIS tag, potentially making it unavailable for the NiNTA resin in the properly folded protein. Have you checked whether you get the same (i.e., disappointing) result with the tag located at either site? Also, do scan your purification protocol for absence of any potential protein aggregation steps. Finally, can you be sure your detected 47kDa is not simply your 41kDa post-translationally decorated or differentially truncated, e.g. due to heterologous expression environment? (protease digestion; MS analyses).

Hello Amit, 

Yeah, this is an integral membrane protein, so I have used 8M urea. Previously protein was visible in cell debris, then I tried for 8M urea, now I got my protein in soluble form, protein and visible in lysate. Then I performed the purification, even after coming in soluble form, Is there any chance of protein to coagulate? As you mentioned, denaturing condition will be the only way, but this condition may degrade the His-tag from the protein, so I am trying it in 4C. I will try to perform binding @ room temperature. 

Hello Marcin,

No I haven't checked that. I will look for that. You raised a very good point. This protein usually goes under post-translational modification. 

Dear Priyanka,

Urea is no way going to degrade your protein. Also any protease is rarely going to be in active form in buffer with 8M urea. My suggestion would be not to go to purify at room temperature as it would reduce stability and any chance of subsequent re-folding. I would rather ask you to go for gradual dialysis with which I have got much better result with an insect protein. Also if you, by chance, have any doubt about the cloning strategies or primer design, make sure that the HIS-TAG is expressing right by doing a western-blot of the expressed protein (with the lysate supernatant).

Good luck,

Thankyou Amit for the valuable suggestion.

You are a linear elution or gradient elution?

It is likely your protein is forming inclusion bodies when expressed. I have developed a protein purification protocol found here http://www.ncbi.nlm.nih.gov/pubmed/20199576 to deal with inclusion bodies and it worked well for a couple of membrane proteins. The key is spiking wash and elution buffers with less than 0.3% sarkosyl. Membrane proteins tend to precipitate out on the column and results in little to no purified protein. Proteins can be dialyzed against your favorite buffer, but it is hard to remove the sarkosyl if that would have any impact on your research.

Just to clarify, your protein is sticking to the column and elutes at 250 mM imidazole, it is just not totally pure. Is this correct?  Or is it after solublization you only have 4 proteins?  Either way, If you have only 3 other bands (even a nickle column) it is not that bad.  

I would try an ion exchange column. Find the PIof your protein and determine what charge it has at the pH you are using. Then select an cation exchanger or anion exchanger accordingly. 

1) change the tag position 

2) clone a Strep tag along with the his tag and try N and C term and try TAP purification

3) have the proteins identified to be absolutely sure that they are degradation products

4) if you are able to refold the protein, then ion exchange chromatography or HIC should separate those proteins.

5) Add AEBSF as a precaution and as mentioned above try at 4C

6) try Guanidine HCl instead of urea for solubilization 

Dear Priyanka,

The anti-his-tag antibodies are usually cheap, please ask someone in the laboratories around for a small portion. For the visible protein you use it at the dilution 1:100 000, this western can be informative for you as well.

Keep fingers cross - jan

Dear Priyanka,

I purified some proteins in 8M urea and the His-Tag worked correctly. For the present of other protein after the elution with 500mM imidazole, try a gadient of inidazole before use the 500mM buffer.

best 

riki

You mentioned you had good expression of your protein.  However, are you positive that a decent percentage of your protein is in the soluble fraction after lysis and centrifugation?  It seems that urea may not be enough to denature a protein that is not primarily in the aqueous phase, but protected by the inner membrane (hydrophobic) environment, like an integral membrane protein. This might require including some mild detergents.

I would recommend some microscale purifications followed by a simple western analysis of all of your various samples along your purification scheme (including whole cells, soluble lysates, insoluble lysate material, resin before and after elution, etc.), as this might help you answer the following: Is your protein soluble? Is your protein binding to your resin?  Is your protein eluting from your resin? And so, on.  This will inform you on the necessary next steps.

Good Luck!

First you must try to optimise conditions of Culture growth and induction etc.Try to induce at lower OD values (.5),  Use .1 mM IPTG final conc. and low temperature post induction 18-20 and pots-induction period should also be shortened.

Check if your protein predominantly goes in soluble fraction or in IB .Also ensure proper sonication.

Second, use high concentration of NaCl ( you can use anything between 350mM to 500mM in binding buffer to minimise protein protein interactions .Do equilibrate Ni-NTA resin with 5-6 CV of binding buffer before loading protein.

Do through washing with Binding buffer before 

i was also facing similar problems. u firrst have to make sure that the gene is in the frame for that u have to get it sequenced. in some cases only the mature peptide comes to fore i.e on the gel or in the purified elute.

do the standard

if ur sequence is ok

take a fresh culture and streak on the plate with kanamycin. take a colony and inoculate it in 5 ml LB broth containing the kana. for overnight  shaking incubation at 220rpm.

take small say 25 ml LB with kana and inoculate with 250 ul of the overnight culture. wait till the OD 600 is 0.6 then induce the culture with 1mMIPTG take 1 ml each after 4, 8 hrs. on parallel take say 5 ml inoculated culture and dont add iptg in it. take cultures on the intervals already mentioned 

adjust the cultures to equal ods i.e say the od at 4 hrs is 2.5 it is but natural that the od 0f culture at 8 hrs will be greater thus add simple broth to the the culture (1ml) u took at 8hrs interval to level it with what was at 4hrs. then run at 12% page. 4 protein samples 

1. non induced 4hrs

2. Induced 4hs 

3 NI 8hs

4. I 8hrs

if ur protein appears that is well and good. 

u dont proceed to purification from a higher to lower conc start from lower to higher

refer to the Ni-Nta manufacturers manual what they have recommended 

usually for lysis 5 mM imid

wash 20mm

elution use gradient 100, 150,200, 250mm proceed inshaAllah every thing will be alright All the best