Скачать или смотреть Gradient elution strategies for protein chromatography (esp. His-tag chromatography via Ni-affinity)

Gradient elution strategies for protein chromatography

“Elution” is basically just a fancy word for “un-bind” or “come off.” You frequently see it in the context of chromatography as well as various solid phase extraction methods like silica-based micro spin columns for nucleic acid purifications (such as PCR clean up kits). What these methods have in column is that you get a molecule of interest to bind to a solid phase (e.g. resin (little beads) in a chromatography column or the silica membrane in a spin column) while letting the rest flow through; then you wash off the weakly- bound stuff; and finally, you kick off your stuff. And this last part is the elution.

more on elution: blog: https://bit.ly/elution ; YouTube:    • Elution terms, strategies (stepwise, gradi...  

You usually* get something to elute by changing the mobile phase (the liquid moving through the solid phase) - such as by using a buffer containing a high concentration of a competitor molecule that will compete for binding sites, or by changing the salt concentration, pH, polarity, etc. This solvent you use to elute your molecule of interest is called the eluent, and the solution containing your molecule of interest that comes out is called the eluant (but we typically just call it the elution).

There are exceptions including isocratic elutions, where the buffer concentration is constant (you see this in size exclusion chromatography (SEC), aka gel filtration, where the molecules don’t bind to the beads but instead travel through them at different rates and thus come out the base of the column (which we still refer to as elution) at different times.

Another exception is if you do an on-column cleavage where you have a tagged molecule attached through the tag to the solid phase (such as a His tag bound to Ni-NTA) and then you cut off the tag, freeing the protein to elute.

When you do an elution, you have options. You can do stepwise or gradient.

A few benefits of stepwise are:
you can do it easily with gravity flow
you typically elute in a lower volume because all your sample comes out at once
You know the concentration of stuff in the eluate

Benefits of gradient are:
you can use it even if you don’t know what concentration is needed to get your sample to elute
You can get better resolution between things that are moderately-strongly bound and strongly-bound

Downsides of gradient:
harder to do without an HPLC or FPLC machine (e.g. an AKTA)
Your molecules might elute over a wide volume (broad peak on chromatograph)
Might have to concentrate a lot
Won’t know the actual final concentration of the buffer/solvent because you had to pool fractions from over a range of concentrations

A sort of in-between option is to do a stepwise elution with multiple steps. This way you can hopefully prevent moderately-tightly bound molecules from eluting with your molecule of interest when you aren’t sure how high a concentration you can safely wash with.

Regardless of the method, you typically do the binding and washing at low competitor concentration. But not no competitor. You want a little in there to prevent non-specific binding so you don’t have random stuff sticking and hogging space that you then have to go un-stick. Better to prevent it sticking in the first place, when there’s still your molecule around to bind instead.

Also, you don’t want to go too low with salt concentrations or you risk crashing out (precipitating). More on that here: blog: https://bit.ly/crashingout ; YouTube:    • Practical tips for preventing proteins fro...  

More on ion exchange chromatography: http://bit.ly/ionexchangechromatography &    • Ion exchange chromatography protein purifi...  

More on immobilized metal affinity chromatography (IMAC) such as Ni-NTA: http://bit.ly/histidineimac

more on size exclusion chromatography (gel filtration): http://bit.ly/sizeexclusionchromatogr...

more on interpreting chromatograms: https://bit.ly/chromatograms

more on fractionation: https://bit.ly/fraction_collecting

more on nucleic acid spin columns: http://bit.ly/spincolumns

lots more on protein purification: http://bit.ly/proteinpurificationtech &    • Protein chromatography  

more practical lab tips & tricks: https://bit.ly/lab_tricks_page &    • Practical lab tips & tricks    

more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com                               

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