Follow the protocol from the manufacturer of the imac gel. Imac is a pseudo-affinity technique; thus it is rarely used as a single purification technique. If many contaminant proteins were removed by heat treatment, use the supernatant fraction to run imac.

Thanks Omar Gonzalez-Ortega for your advice ,but can you suggest possible buffers with their concentrations that I can use for the purification , since the resin kit that I want to use did not come with buffers , so I have to prepare them on my own. Can I get a good compositions for the purification buffers?

Simply titrate from 0 to 0,5 M of imidazole solutions (in alog scale) and elute until you find the correct concentation for optimal elution. I would use a safe low concentation (1/100 of the elution concentation) of imidazole for washing. The optimal imidazole elution concentaion depends on the HisTag and molecular weigh of your protein.

Sodium phophate or phosphate citrate buffer preferred over tris, but tris can be used successfully

Here you go :

1) Equilibrate column using 20mM Sodium phophate buffer, 0.1M  to 0.5M Nacl, 10mM imidazole

2) Wash : Same as equilibration buffer ( at least 10 cv )

3) Elution : 40mM imidazole, 100mM imidazole, 200 mM imidazole, 300mM imidazole, 500mM imidazole ( You have to test various imidazole concentration, run gel and wstern to find where you get super high yield, except previous papers of same protein no one can tell you where your protein will elute )

Columns can tolerate 1mM TCEP, 10mM DTT, and 20 mM BME , however you have to wash columns immediately after use ( Dont store column with these, and you will never use this high end concentrations of DTT, BME- 1/4 th concentration of these should be plenty) 

1mM EDTA, EGTA can be used in rare case, but usually I avoid using these chelator.

Good luck !!!

Dear Suleiman

generally i'm using

lysis/binding buffer:

tris 20mM , Nacl 300 mM imidazole 10mM pH=8

wash buffer

tris 20mM, NaCl 300 mM, imidazole 20mM ph=8

elution buffer

tris 20mM, Nacl 300 mM , imidazole 300mM ph=8

to increase the purity level of you protein i suggest to you:

1) after protein loading and ft collection wash a lot with binding buffer ( in small imac i use also 60-100 cv)

2) of course if you can use an akta sistem you can perform a gradient from the binding to the elute.. but it have to be very slow.

3) try to minimize the resin volume to be close to the saturation because ifyou use to high resin amount your protein is not able to saturate all the nickel and more impurities will attach, if you are close to the saturation your protein will compete with the impurities and the product will come out more pure.

good luck

Manuele

@Manuele Martinelli, thanks very much for your suggestions. I will try those buffers and conditions suggested.

the best way for purifing recombinant His-tagged proteins that I have used so far is the protocol that I attached here for you.

you should use the part of native buffers in pages 13-14

I have achieved bacterial proteins with high purity by using this protocol.

The best protocol you can use is the one that you generate after you understand the principle of the HisTag and imidazole buffers. The optimal buffer molarity for the elusion will vary according to the molecular weight, polarity and structure of your protein and other factors. Sometimes the difference is negligible when using 100 to 250 mM imidazole for elusion but if you are unlucky the pK for your complexes could be beyond these values. So I would recommend trying different buffers systemically until you find the optimal one.