I have used an Ni-NTA system for purify five different his tagged proteins produced in E.coli , optimizing the amount of imidazole for the equilibration, washing and elution steps however the purity of the protein isn´t more than 80%. The unespecific bands visualized by SDS-PAGE are bacterial proteins that bind to nickel, and these are the same for all the lysates purified. I have seen protocols for his-tagged proteins purification that include two steps more of purification: anionic Exchange and heparin-trap chromatography columns. As I don´t have a FPLC equipment I wonder if the Pierce Strong Ion Exchange Spin Columns might work well for this purpose or if there is another strategy I can try.
It is quite normal that an affinity chromatography for a protein tag (like His6) does not give you 100% purity. However, it is still a great capture tool: Your target may go from, say, 1% in the cytosol to 80% in the eluate. This means you can use small columns with high resolution (= expensive) media for further purification. High resolution means small beads means high back-pressure, so a make-shift setup with a two-chamber gradient maker and a peristaltic pump may not work too well.
The spin columns can replace an FPLC system up to a point. In place of a chromatographic pump, you use centrifugal force to drive fluid through the column:
The one thing you cannot do with these columns is to use continuous gradients (I did say "up to a point"). The best you can do is use step gradients, then each step is a separate spin. Especially for method development this can be tedious. My advice would be to negotiate access to an FPLC system from another lab, if that is possible. Of course, that depends on how often you want to do a purification, and how busy the machine is. In some places, you may have a core facility where protein purification and characterisation is performed.
For heparin, you can have reasonably selective binding, so spin column might be fine. For ion-exchange I'm guessing you probably want gradient capability and a reasonably high quality column packing, with a few cm column length, to achieve effective separations of your impure fractions, depending on what all the isoelectric points end up being.
"FPLC" is just a way of saying pump + detector, in a fancy housing. You can drive a real FPLC column with syringes operated by hand, if necessary. If you prepare several syringes with pre-made salt concentration, you can have something approximating a gradient. You can detect protein in the fractions by UV spec or BCA. This is not actually a disastrous amount of work if you get the hang of it. I used to do some very successful purifications in this way, before I had any money.
Alternatively, you can probably find some kind of pump at your lab. Two syringe pumps + inexpensive check valves make a continuous flow device. Or perhaps you can find a small peristaltic pump. Anything that can get a small stream of liquid moving at a few PSI can do the trick. Then you can make a gradient with a cheap chemist's gradient maker glassware device.
Dear Angeles,
I would simply bind the protein in bulk to the resin after a buffer exchange, remove the resin, and do stepwise elution by incubating with increasing amounts of salt. It is the cheapest way and beats spending money on columns.
Spin-through columns could be made by yourself. Take an old cartridge/pipette, put some glass wool inside, cap it with another layer of wool, and use gentle centrifugation at 500-1000xg for 5 min max. Biorad used to have tiny columns for gravity with built-in frits and you could reuse them.
Wes
Hi,
After NI-NTA purification, you should choose an additional purification step we can also referred to as a polishing step. If your SDS-PAGE visualization gave that MWs of the proteins are not very closer to each other and well seperated rather than closer bands than I can strongly suggest the size exclusion chromatography (SEC) due to it could help to remove some of the degraded protein from the intact and also other interfered host cell proteins. You may supply the Gel Filtration bulk resins like commercial brands Sephacryl / Sephadex/ Sepharose / Superdex / Superose /Toyopearl and make your own column chromatography depending for your application details.
Dear İsmail Emir Akyildiz , Wieslaw Swietnicki , John Schloendorn , Engelbert Buxbaum , thank you very much for so interesting suggestions you have made to me. I´ll try to have access to an FPLC system but if ithat is not possible I will test the home made systems.
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