Hi there

Before doing the chromatography, please check by SDS-PAGE if you can see the band of your protein after induction. If not, you can try a batch purification with a small amount of the Ni-resin to see if you can enrich your protein. Do not forget to perform the experiment using the soluble and insoluble fractions. Maybe you are not seeing your protein because it is insoluble.

I do not think that you could have problems with the buffer. Everything with pH between 6 and 7.5 should work. I use to employ a high salt concentration to avoid cross-contamination.

Good luck

Hi there,

As mentioned above, first check the expression level and the solubility of your target protein. Then check the presence of the tag by WB using anti-His antibody. Then check every fraction of your purification by SDS/PAGE and WB.

If the protein is expressed then the basic issues are :

accessibility of the tag : in that case the protein goes into the flow through (to check this run SDS/PAGE) If itis the case then repeat the loading step with prior addition of a non denaturating concentration of urea or detergent to the sample and see.

protein remains stuck to the matrix (aggregation or unefficient competition of imidazole). To check this, put a small amount of matrix in SDS/PAGE loading buffer, boil it and load onto gel.

About buffer composition, I would be more strict than above: pH has to be above 7 because both histidines of the tag and imidazole used for elution have to be vastly protonated to interact with Immobilized ions of the matrix (efficient interaction with the matrix imposes this). As the pKa of both is somewhere between 6.5 and 7, pH 7.5 is the minimal value required for all the buffers.

Hi there,

Your gel is not very clear about the nature of the samples you have loaded and also because some lanes are overloaded (lysate, control) and contaminates lanes next to them... What are the steps and conditions of your purification? How much imidazole do you use for washing and elution? What you should repeat is running a gel with descent amount of proteins in each lane so that you can actually follow the purification (uninduced culture, induced culture, soluble lysate, insoluble lysate, flow through(ie. what goes through the column without being retained, washes at low imidazole concentration (at least 2...), elution steps (several with an increasing concentration of imidazole, final elution using edta and finally a resin sample in case the protein sticks to it).

Yes you need to rerun your gel using at least a 10X dilution of the samples in lanes 4 and 9. The samples in lanes 1,2, 3 and 8 could be reduced by half as well. In general you should use ~ 5-10 ug of protein per well, less if its highly purified and mainly in one band. Examples of some lysates from uninduced and induced cells from our lab is attached.

Thanks all

I can’t repeat it

but your comment was really useful .

Checking for proper induction and solubilisation of the target protein by electrophoresis as already mentioned is useful.

Another important factor is the protein concentration. If this was determined only by A280 then other UV-absorbing compounds may have caused too high a reading.

Thirdly, you should check that your target protein actually binds to the IMAC column, the flow-through and wash should no longer contain it (also no "break-through" towards the end due to overloading).

Finally, the protein should be washed of the column with elution buffer.

Keep book about your purification: note the volume, total protein concentration and concentration of target protein for all fractions, so you can calculate recovery and purification factor for each purification step.

Oh, and please: check spelling and grammar before you press send (if your native language is not English, use appropriate computer programs).