If you are seeing bands in the FT, it could be that you are not using enough resin for the amount of lysate you have. As such, your "desired protein" is found in the FT. So I'd look at that first. What is the binding capacity of the particular resin, and are you loading an appropriate amount of sample?

As for timing, most of these interactions are fast and I would expect 5-10 min would be the max people are using. I'm assuming here you are not using these resins in an online system, but offline in a type of "Stage tip" setup, or in an eppendorf tube. Again, check protocols in the literature and see what timings others are using.

Having said 5-10 min, I just double checked our IMAC protocol and we incubate for 30 min during the binding step. Just to confirm, this is for complex samples and is performed in an eppendorf tube. Timings are likely to vary with sample complexity and no doubt will be different for other resins.

Hope that is of some assistance,

Peter

Hey Peter,

Thanks for your suggestion. Actually I incubate the lysate with resin in falcon tubes for around 15 to 20 min at 4 degree C with constant rotation (to help resin-protein binding).

You are right. The question was mainly concerned with Batch-mode of purification. I generally take resin to lysate ratio around 1:10. I guess that should be enough.

Thanks for the answer.

Sagar Sridhara

You may be seeing your protein in the FT because the 6-His tag is being clipped off by proteases. Make sure you add a non-EDTA containing protease inhibitor tablet to your lysate. The capacity of IMAC resin is pretty high, so it is actually difficult to saturate the resin unless you have incredible expression levels.

It takes nearly 15-20 minutes, further you have to be quick. However, I will suggest to use column chromatography approach. Rather to use in batch, you may try it.

Resign:Lysate ratio of 1:10 may not be sufficient, as (i) resign may not have sufficient capacity and (ii) even if it does (if you are calculating expected amount of protein, discharging others) there are plenty of other protein that do have HIS-tag in them, but usually with lower affinity to IMAC, that could block resign. Plus, your's redign "matrix" could bind "impurities" via anion exange.

To overcome these, I usually add NaCl up to 300mM to binding buffer or to resign, (to block "anionexange" interactions), and if, possible, I use sulphate precipitation to increase amount of protein of my interest in lysate.

I guess Denis was referring to undesired proteins with His-tag.

Proteins such as chaperon(SlyB) has Histidine repeats with around 10 His on their C-terminal sequence(I guess), they strongly bind with Ni/any divalent ions!

Maybe plenty was a too strong word I used. I am sorry for that, prof. Zoran Vujcic.

Sagar, are we talking about bacteria cell lysate? As if you use lysozyme or DNA hydrolase for preparation of cell lysate, you should pay attention for its' origin. If it is recombinant, it is highly possible that it has HIS tag on it, so it will bind to IMAC. I once had this situation.

The bacterial cells were expressing a recombinant human protein in TB media. So, the cell lysate will have both bacterial proteins and the desired recombinant ones.

Aiming to improve the binding of protein in any chromatographic matrice I think that the crude preparation must be decreased in your complexity. So, I suggest you to submit, for example, the lysate to ultrafiltration in 10 kDa cut off membrane, ammonium sulphate or acetone (50% v/v) precipitation followed in the first case of dialysis. Decreasing the molecules complexity you will ensure a better interaction of the proteins/target protein(s) in the stationary phase of the chromatography.

What happen if we incubate Lysate and resign mixture over night

It depends upon the kind of Tag you are using for protein purification.

Generally His TAG binds much faster in comparison to other tags.

Heparin works as ion exchanger so, max. 30 mins incubation with cell lysate at 4C is sufficient.

In the case of Streptavidin Magnetic Beads, I often incubate with protein and rotate the tube overnight. The recovery rate is higher than incubation with one hour.