What is the expected size of your protein ? If it is bigger than 23 kDa, it is quite possible that the 23 kDa band is a fragment of your protein that also bears the His-tag. In this case, you will probably have to use conventional purification methods to get rid of it, e.g. gel filtration if the target protein is sufficiently bigger. Otherwise, you could try to increase the imidazole concentration in the wash buffer. With Ni-NTA, it is advisable to use at least 15 mM imidazole pH 8 to get rid of non-specific bands. When the tag of the target protein is well exposed, it is sometimes even possible to wash with 35 mM imidazole, but it is advisable to check the fractions by SDS-PAGE.

hi,

my protein size is 12.8 KD and I tried with 10mM, 20mM, 50mM and 70mM Imidazole in rinse buffer (I use last portion of wash buffer as rinse buffer because I change the detergent from SDS to LMPG) 10mM, 20mM did not work I still had that protein, but in 50mM and 70mM I loose lots of protein of my interest, since I work with small membrane protein It absorb lots of detergent so, it is hard to separate using gel filtration.

Thanks

Did you tried further purification by gel filtration or ion exchange? you dont always get >95 % clean protein after NiNta. Depending on the protein and expression level, i had both situation. while in few instance, i got very clean protein after ninta that there was no need to do any further purification. some time i had to do 2-3 extra purification steps to get clean protein.

Hello

In my opinion, eventually you can get pure target protein by use the Reverse-Staining on large scale SDS-PAGE gel ( Imidazole, Zncl2) and isolating the target band from the gel and then extracting it from the gel by electroelution method, and get rid of extra protein bands.

Good luck

Hi Dhiraj,

I did not try gel filtration, I will try that. Thank you for your suggestion

Hi Touraj,

Thank you for your answer

Hi Fathima,

Your welcome

best

If the 23 kDa band is in your induced cells but not in your uninduced cells on SDS-PAGE, then it is likely a proteolytic fragment or maybe it is a secondary start site on your gene. Check your gene and see if their is a AUG downstream of your translation start codon. If it is proteolysis, make sure you are using protease inhibitors when you lyse the cells (ie. PMSF). If these two reasons are not the causes, then perform an IMAC purification and see if it is present afterwards. If it survives the IMAC purification, then it must have the his-tag.

Hi, I checked my sequence it has ATG in the middle but at the end I have stop codon so, even the translation starts at the middle it should give small size protein band in SDS-PAGE not a big one. my problem is I have bigger band than the target and it can be easily eluted with Ni-NTA and it binds stronger than my protein of interest

OK, if I understand correctly, this protein is larger than your protein. I have a couple of suggestions. (1) check your expression and lysis to see if your getting the maximum amount of soluble protein. I have had cases where Ni NTA purification comes out impure due to low amounts of soluble protein after lysis. (2) If this is the best purity you can get, use a second ion-exchange column after an overnight dialysis. It is very unlikely that this protein will have a similar pI as your protein as well as bind to the Ni NTA column.

Usually the NiNTA is used for the initial purification of proteins. Most of the times the eluted fractions contain also additional proteins. If you get your protein nicely overexpressed, its binding is more preferred and you might get cleaner fractions. Moreover, you can add some imidazole (10-20 mM or so) into your protein solution before carrying it on the NiNTA column - it might reduce the binding of irrelevant proteins. I am not sure how the rinsing buffer with Imidazole mimics this procedure. Thirdly, sometimes it is good to use an approach when you increase the imidazole concentration stepwise or gradually - for this you need an automated system (AKTA etc). At certain imidazole concentrations you might get better results.

In your case, I recommend to use additional purification steps (ion exchange, gel filtration) to get your protein >95% pure. Sometimes you need to repeat a purifictaion step multiple times. I am sure that there are several protocols for membrane proteins which can help you prepare buffers and equilibrate columns with the preferred detergent (we usually use two SEC columns - one is detergent-free and the other is for detergent-dependent proteins).