Batch binding with gravity flow chromatography

I love FPLC (Fast Protein Liquid Chromatography) - but sometimes, I still feel gravity flow is the way to go! (And, thankfully, my students found it a lot of fun!) This is especially true if you have large volumes, lots of protein, and/or viscous samples. Although you can control many things more precisely with the FPLC, you can customize more and easily adapt on the fly with gravity flow. So, I often prefer to do affinity chromatography using good ‘ole gravity flow (with batch binding which I’ll talk about below) before moving on to the fancy machine. But, speaking of moving on, let me start by stepping back and providing some background . . . https://thebumblingbiochemist.com/365-days-of-science/batchbind/ In column chromatography, you flow liquids containing molecules such as proteins traveling through cylinders filled with little beads called resin. Different types of resin have different properties, and different proteins have different properties. Therefore different proteins will interact differently with different types of resin as they flow through the column. And this allows us to separate proteins based on their properties. One of the main types of column chromatography used for protein purification is affinity chromatography (sometimes referred to as affinity capture). This takes advantage of a unique feature of a protein of interest to get it to bind specifically to the column while other proteins don’t bind (or, if they do bind weakly, they can easily be washed off). Often these “unique features” are affinity tags placed (based on adding genetic instructions) onto the ends of the proteins. These tags (His tag, strep tag, FLAG tag, etc.) are simply short sequences of amino acids that will bind specifically to some kind of resin. In the case of His-tags (a string of 6-8 histidines), they’ll bind to nickel-coating columns (commonly Ni-NTA, where “NTA” is a linker molecule used to attach Ni to the beads. A solution (such as a cellular lysate – the inside content of cells) containing a His-tagged protein of interest along with other unwanted molecules can be flowed through a Ni-NTA column and the protein of interest will stick but the others won’t. Some proteins might bind weakly (through “non-specific” binding), but they can easily be washed off while the protein of interest remains firmly stuck. Eventually, however, you want the protein of interest to come off (elute). To get it off, you can push it off through competition with something that looks like the tag. In the case of His-tags, the competitor used is imidazole, which is basically the isolated ring portion of histidine. By adding a lot of imidazole, you can get your protein to come off – now without all those contaminating proteins! So, the basic strategy is: 1. Equilibrate column (get it ready to bind) 1. Buffer used has low concentration of competitor to prevent super non-specific binding 2. Bind 1. Buffer used has low concentration of competitor to prevent super non-specific binding 3. Wash 1. Buffer used has higher, but still low, concentration of competitor to wash off proteins bound weakly through non-specific binding 2. Wash until protein stops coming off (can determine via Bradford or UV280 absorbance) 4. Elute 1. Buffer used has high but concentration of competitor to kick off the protein of interest 2. Elute until protein stops coming off (can determine via Bradford or UV280 absorbance) All of this can be done in an automated manner using an FPLC (Fast Protein Liquid Chromatography) machine such as an AKTA, which pumps liquids through pre-packed columns*. Or, it can be done using gravity flow, relying on gravity to pull solutions through manually packed columns. *There are also empty columns you can fill yourself. When I do gravity flow chromatography, I like to do most of it in the column format, but I prefer to do the binding step in “batch mode” – basically just mix the protein-containing solution with the beds in a tube (e.g., a 50-mL conical tube), giving the beads and protein time to meet one another. To do this, just add the solution to the resin in the tube and place in a cold room on a tube rotator for 30 min-1 h. Then, transfer back to the column. The resin will resettle into a column and you can proceed with your purification protocol. I’ve found you can get better binding this way than just letting things flow through and relying on the protein and resin meeting each other and having enough “oh hi!” time to bind and stay stuck. Finished in comments