The classical solution would be to perform a gel filtration after the Ni-NTA. Usually, the size exclusion cromatography should separate your protein from the high and low MW contaminants. There are another solutions, but firstly I would try the gel filtration.

The classical solution would be to perform a gel filtration after the Ni-NTA. Usually, the size exclusion cromatography should separate your protein from the high and low MW contaminants. There are another solutions, but firstly I would try the gel filtration.

I agree with Jaime that you should add another purification step. It could be gel filtration or, after reducing the salt concentration, ion exchange chromatography.

However, there may still be room for improvement in the Ni-NTA purification. If you are getting a lot of the desired protein in the flow-through, there are several possible reasons: (1) The column is overloaded. You need to load less extract or use a bigger column. (2) Some of the protein has the His tag cleaved off by protease. That protein will be lost unless you can reduce proteolysis. (3) Some of the protein is aggregated, and aggregated protein doesn't bind to the column. The extraction procedure or buffer may be changed to reduce aggregation. A lower salt concentration might help.

Thanks for your suggestion guys, with our current lab facility it wont be possible to go for second step purification with gel filtration or ion exchange, so is there any other option available.

Agree with Adam that there is room for improvement in the Ni-NTA purification. Increasing the imidazole concentration during binding and washing should improve protein purity although you may also get some loses of your protein. We make use of ReSyn Ni-NTA beads (see link below) and use 80mM imidazole during binding /washing. The high functional density of these beads allows use of higher imidazole conc without significant loss of target protein. Also, since the beads are magnetic the purification can be automated if you have access to a magnetic separation station such as the Kingfisher. See last link for an example where phosphopeptide enrichment was automated using magnetic TiO2 and TI-IMAC. 

Increasing the salt conc to 0.5 M can reduce non specific interactions and thus also improve purity but as mentioned above this may also lead to aggregates in some cases due to salting out effect. 

http://www.resynbio.com/nta.htm

http://www.thermoscientific.com/content/tfs/en/product/kingfisher-magnetic-particle-processors.html

http://pubs.acs.org/doi/abs/10.1021/ac5025842

How about ammonium sulfate fractionation? Proteins precipitate at characteristic ammonium sulfate concentrations, so this method can be used as a purification step. It requires only a magnetic stirrer, a cold room or cold box, and a centrifuge. Ammonium sulfate is a very non-chaotropic salt, so it should not denature your protein. (NaCl, in contrast, is somewhat chaotropic and should not be used instead).

In the cold, slowly add crystalline ammonium sulfate to 10%, 20%, 30%, etc, of saturation (there are tables to help with the calculation). After each step, allow some time for the precipitation of proteins, then centrifuge to recover the pellet containing whatever proteins precipitated at that concentration of ammonium sulfate. Continue adding more ammonium sulfate to the supernatant.

If you have an ultracentrifuge with a swinging bucket rotor, you can use sucrose gradient centrifugation as a purification step.

You don't need fancy equipment like an FPLC to do gel filtration or ion exchange chromatography. You can use an open-top glass column (such as Bio-Rad Econo columns) and fill it yourself with the resin. You can collect fractions by hand. Salt gradients can be formed with two beakers, a magnetic stirrer, and some Tygon tubing.

I have found that charging the column with Cobalt instead of Nickel leads to a cleaner elution.

 Also a 12His tag allows you to increase the amount of imidazole in the buffer and will also increase binding of your protein to column - perhaps the tag is inaccessible and that is why you are getting your protein in the flow-thru (a linker such as the TEV protease cleavage site or changing the termini of the His-tag may help with this). 

Hi,

I noticed there is lots of target protein in flow thru. (1) It could be the problem of column volume. The solution is to increase volume of column and load three times to see whether the quantity will be increased as well. (2) If the solution (1) doesn’t work. It indicates some of the protein may not be folded very well. If there is a way to slow the production of this protein, more well-folded protein could be acquired. I hope it is helpful.

How about lowering serum concentration in Graces insect media ?? I am infecting SF-9 adherent cells with baculovirus in media containing 10%FBS. So Shall I reduce the FBS to 3% or even less to slow down the protein production if thats the case with folding, Will that work??  Please look into the gel pic.

Is most of your protein correctly folded? If not, probably it will be very difficult to get rid of chaperones that are contaminating the sample, because these are attached to your polypeptide.  Try to remove chaperones (see our work http://www.ncbi.nlm.nih.gov/pubmed/12182832)

In many cases it is good not be too ambitious with the purification and just collect the appropriate fractions helps. Another important issue is generating the lower denaturation of proteins during cell lysis. More denatured protein from the beginning more contaminating proteins  in the enzyme to be purified.

Before we do anything about the folding, you may need to check whether it is correctly folded. i would suggest you denature the target protein from flow thru and then reload it on column to see whether it bind well or not. if it does bind, you could try to change the temperature of expression.

I think purity from ni sepharose is not high, so you should use ion exchange or gel filtration chromatography after that. And as Yong Li is mentioned, sometimes protein aggregates because of misfolding and it inhibits the binding of the protein to resin. I think gel filtration is good way to check the aggregation. And if there is cleavage site between target protein and his-tag you should check that protein can be digested by protease, Aggregation also inhibits the cleavage.

I suppose this is a recombinant protein, is using a gst tag in spectrum of possibilities.

Thanks Eduardo for the reply. I have few questions:

1. The article is more with the E.coli as host for protein expression, so will it translate similar way in insect system?

2. Which chaperons are expressed in case of insect cells ?

3. Morover phosophorylation status of my protein is also one of the important factor to be considered which may limit the use of ATP in purification process.

If anyone can share the detail protocol for gel filtration chromatography using glass columns( for gravity flow), right from column preparation to elution.

The Hsp70 chaperones, although they could have some peculiarities in specificity, they are present in the cytoplasm of eukaryotic cells. There is evidence that some viral proteins that interact with Hsc70 cytoplasm of fibroblasts, also do it with a Hsc70-like protein from baculovirus and with DnaK (Hsp70 from E. coli). Therefore, if the contaminants are of approximately 70 kDa, they could be Hsp70s. Regarding point 3, I do not have a solution for that problem.