I have frequently found precipitant when dialyzing imidazole out of the buffer. Very little of my protein was in the precipitant therefore it is usually just the imidazole itself. Have you checked if you loose protein during the dialysis step?

As for the enzyme activity, it could be that keeping the protein in imidazole buffer for two days harms the protein. Also, how do you keep the protein for those two days? It would probably be best to complete the purification as quick as possible then store the enzyme in a more suitable buffer and get it frozen away.

I have frequently found precipitant when dialyzing imidazole out of the buffer. Very little of my protein was in the precipitant therefore it is usually just the imidazole itself. Have you checked if you loose protein during the dialysis step?

As for the enzyme activity, it could be that keeping the protein in imidazole buffer for two days harms the protein. Also, how do you keep the protein for those two days? It would probably be best to complete the purification as quick as possible then store the enzyme in a more suitable buffer and get it frozen away.

Hi Fereshteh,

Generally The activity of Enzyme gradually losses in a buffer containing imidazole for more than 24 hours. So, its better to dialysis the protein or enzyme less than 24 hours after purification to remove the imidazole . Where as the imidazole is of size 0.68KDa so its better to take the dialysis bag having less cut off than your protein and more cut off than the imidazole. Dialysis buffer composition should be the same buffer which the protein was eluted but concentration should be less.

All the best

hello Christopher

thanks for your answer

how can I check that my protein is lost during dialysis?

after purification I immediately keep my enzyme in -20

when I wanted to run my enzyme after dialysis on SDS-PAGE, I found the white participate at the bottom of microtube ( after boiling with dye for 5 min).

when i compare the sharpness of two bands ( before and after dialysis), the band of dialysed enzyme is weaker.

Which kind of enzimatic activity as your protein?

Which are the differences between the elution buffer and dialisis buffer? just imidazole concentration? What aoubt salt? pH?

in my opinion you can certanilly try to speed up the buffer exchange procedure and use desalting (that is a fast procedure, in place of dyalisis). you can concentrate the elute of imac coloumn by amicon ultra centrifugal concentrator and load your sample in a PD-10 desalting coloumn (Ge) equilibrated with the new buffer. in 10 minutes you will have the protein in the new buffer.

i use often this protocoll after elution from imac to put the protein in buffer for TEV digestion and in my experiences it works quite well. Of course if the problem is the intrinsic stability of your protein, this will not solve the problem.

good luck

hello Manuele

what do you mean which kind od enzimatic activity?

my enzyme is cellulase, I remove small domain from it. it's themozyme enzyme. I don't think it's sensitive to 4 C.

my dialysis buffer and elution buffer is the same, but I have NaCl and imidazole just in elution. the dialysis ph= 7.4 and elution ph= 8

I can just do dialysis for exchange of buffer

just for undertand which kind of residues are involved in your protein activity.

Has your protein free cysteines? Is it possible that aggregation happen due to cystein ossidation and intramolecular S-S formation?

because in that cases is possible that your protein is produced with free cisteines and during the time the air induce their oxidation and aspecific S-S formation.

try to load the precipitate in the SDS page with and with.out reducing agent and check if you observe a difference in MW. In that case you can try to repeat your purification but mantaining or reducing agent as DTT or TECP.

if you do not have cisteine, i think try the pd-10 to speed up the buffer exchange is a good alternative trials. Which is the pI of your protein?

Hi Fereshteh,

You are saying that you have done the dialysis using the pH : 7.4 and elution Buffer pH is 8. Do you have any idea about the PI of your protein? dialysis buffer Should be more or less than protein PI at least 0.5. all the best.

the enzyme structure has 2 cys that bind to Zn, maybe Zn was removed during purification. cys is n't involved in active site ( Glu and Asp are involved.

What does observing a difference in MW show?

thanks for your guide

hi Ramesh

I can't find any information about PI of my enzyme. but I calculate it by online software, It's about 6.5

it's interesting for me that why dialysis buffer should be more or less than protein PI?

TNX

Hi Fereshteh,

The dialysis Buffer must be more or less than PI. If the pH of buffer is equal to the PI of your protein than, the charges on the protein becomes neutral and this creates salting out environment hence the protein will precipitates. For safety purpose the pH of buffers used for purification or dialysis or for preserving the protein or enzyme activity reactions will be more or less at least 0.5.

the differences in MW in presence and absence of reducing agent may suggest you the possibility that cysteine instead binding the Zinc, are becoming oxidated and produce intramolecular S-S bond.

If zinc is not binded and the cys are free is it possible, expecially at high concentration that the cys of one molecule react with the cys of one other, is it possible that you have aspecific formation of dimer or oligomers, and you will se in the SDS page.

which kind of metal did you used for imac purification ? Ni? Cu? Zn?

regarding the pI, if it is 6.5 is preferable that you keep the pH of your buffer, also the one at the end of dyalisis at 8 instead 7.4, because when you stay to close to pI your protein is not so charged and its solubility in water decrease. You have to consider that 6.5 is theoreticall, and calculation made in linear sequence, but is also possible that your protein may have real pI more close to 7.4

im my opinion you can try different stategy:

1) exchange buffer by pD-10 instead dyalisis using a buffer w/o imidazole with ph=8 instead 7.4

2) check in the gel il precipitate show high mw in assence of reducing agent and if so add a small amount of DTT in the buffer

3) try to change the metal used for imac or add a small amount of EDTA in the elution buffer before dialisis just to prevent possible aggregation due to strip of metal from imac coloumn...

further consideration can be done only on the basis of the result of this experiments.

good luck!

I use Ni

thank a lot for your guide, I try them

best regards

You could add some amounts of BSA if your enz doesn't care for its activity.

Very routine thing seen in case of many protein at a particular ionic concentration. May be zwitterion effect. I usually ignore it as long as they do not interfere with my work.

When you purify His-tagged enzymes with imidazole, the general rule is to remove imidazole ASAP and keep the protein on ice or at 4C all the time. Keeping enzymes in 250 mM imidazole is bad idea regardless the enzyme biophysics properties.

Please check my publication "High yield expression of non-phosphorylated protein tyrosine kinases in insect cells " for a general method to start with. In the publication, it is for a kinase BTK. Your enzyme might be a little different, but it is a good point to start with.