And, just in case, the system has pressure monitors at the entrance and exit of the columns. If the pre-column pressure (pressure going in) is too high it can damage the column hardware (the cylinder itself) & if the delta column pressure (difference between pressure going in & going out) is too high it could the resin in the column and/or the filter on top of the column are clogging up and generating dangerous pressure that can hurt the resin. So the AKTA will stop and alert you. The liquid flows through lots of little tubes that offer different flow paths. Which path the liquid takes is dictated by lots and lots of valves to go with those lots and lots of little tubes. It’s kinda like a subway system that can change the tracks. So we can direct liquid into different columns and, when it comes out of the columns into the waste or a fractionater which collects it to “keep.” We choose which fractions we actually want to keep based on the chromatogram This is where we see the evidence of our protein coming out in the form of a peak in the 280nM wavelength absorbance. Proteins (in particular tryptophan, tyrosine, and phenylalanine) absorb that type of light so you can tell when protein’s elute because they “steal” that wavelength from the light spectrum. A UV monitor on the path between the bottom of the column & the fractionater measures this. And the computer shows this to us as a peak. more here: bit.ly/2yzyi4w FPLC looks a lot like a related technique, HPLC. HPLC stands for High Performance Liquid Chromatography. HPLC uses higher pressures but lower flow rates. It’s usually used for small chemical compounds and sturdier beads that can withstand those high pressures. The kind of chromatography wI use is typically “preparatory” - we separate things to use them in their purer form. There’s also analytical chromatography, where you separate things just to see what’s there. this is similar to one of the differences between SEC & SDS-PAGE bit.ly/2xWH7VZ There are many advantages to using the FPLC over relying on gravity to drip liquid through (gravity flow). My favorite reason - you don’t have to work in the cold room forever because it’s automated and, if you’re lucky, housed in a deli fridge not the cold room. When you’re working with precious proteins, you want to keep them safe. At higher temperatures, proteins can start to degrade especially if there are contaminating proteases (protein chewers) which at cold temps don’t have enough energy to chew your protein but do at higher temps. But I don’t use the AKTA for everything. One of the nice things about gravity flow is that you can pack your own columns. AKTA pre-packed columns come in multiple sizes, but with gravity flow you can choose exactly what you want. I prefer to use gravity flow for initial purification steps, where I’m working with “crude lysate” - the liquid you get when you break open cells (lyse them). Even though you then spin them down really fast (ultracentrifugation) to pellet the membrane pieces and other insoluble stuff and then you push it through a syringe filter, it can still be pretty viscous (syrupy) and this can clog up the AKTA lines. The AKTA’s really smart - it senses the buildup of pressure and slows down the flow rate - so it starts flowing REALLY SLOWLY. So much for that fast part… So it can be much more time-effective to go with gravity flow in these cases. Even when it’s not crude lysate, you still need to worry about stuff clogging the tiny tubes - so we filter all of the buffers we send through them - usually using a bottle-top vacuum filter - it has a kind of “bowl” you screw on to the top of the bottle and you pour the buffer into the bowl and it gets sucked through a membrane into the bottle. Want more details on protein purification and the various forms of chromatography? I have a whole page of posts on my blog: thebumblingbiochemist.com/lets-talk-science/protein-purification/
and a RUclips playlist on protein purification: ruclips.net/p/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7 as well as one specifically on chromatography: ruclips.net/p/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7 more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
Are there more advanced videos? This helped a lot when I started but now I am looking at things like which columns to use and in what order, troubleshooting gradient elutions to better purify proteins, choosing different buffers to try and prevent dimerization etc. Is there anything like that on your channel right now? Thx!
You are a lifesaver!! Whenever I have free time, I always listen to your "podcast"!
Happy to help!
And, just in case, the system has pressure monitors at the entrance and exit of the columns. If the pre-column pressure (pressure going in) is too high it can damage the column hardware (the cylinder itself) & if the delta column pressure (difference between pressure going in & going out) is too high it could the resin in the column and/or the filter on top of the column are clogging up and generating dangerous pressure that can hurt the resin. So the AKTA will stop and alert you.
The liquid flows through lots of little tubes that offer different flow paths. Which path the liquid takes is dictated by lots and lots of valves to go with those lots and lots of little tubes. It’s kinda like a subway system that can change the tracks. So we can direct liquid into different columns and, when it comes out of the columns into the waste or a fractionater which collects it to “keep.”
We choose which fractions we actually want to keep based on the chromatogram This is where we see the evidence of our protein coming out in the form of a peak in the 280nM wavelength absorbance. Proteins (in particular tryptophan, tyrosine, and phenylalanine) absorb that type of light so you can tell when protein’s elute because they “steal” that wavelength from the light spectrum. A UV monitor on the path between the bottom of the column & the fractionater measures this. And the computer shows this to us as a peak. more here: bit.ly/2yzyi4w
FPLC looks a lot like a related technique, HPLC. HPLC stands for High Performance Liquid Chromatography. HPLC uses higher pressures but lower flow rates. It’s usually used for small chemical compounds and sturdier beads that can withstand those high pressures.
The kind of chromatography wI use is typically “preparatory” - we separate things to use them in their purer form. There’s also analytical chromatography, where you separate things just to see what’s there. this is similar to one of the differences between SEC & SDS-PAGE bit.ly/2xWH7VZ
There are many advantages to using the FPLC over relying on gravity to drip liquid through (gravity flow). My favorite reason - you don’t have to work in the cold room forever because it’s automated and, if you’re lucky, housed in a deli fridge not the cold room. When you’re working with precious proteins, you want to keep them safe. At higher temperatures, proteins can start to degrade especially if there are contaminating proteases (protein chewers) which at cold temps don’t have enough energy to chew your protein but do at higher temps.
But I don’t use the AKTA for everything. One of the nice things about gravity flow is that you can pack your own columns. AKTA pre-packed columns come in multiple sizes, but with gravity flow you can choose exactly what you want.
I prefer to use gravity flow for initial purification steps, where I’m working with “crude lysate” - the liquid you get when you break open cells (lyse them). Even though you then spin them down really fast (ultracentrifugation) to pellet the membrane pieces and other insoluble stuff and then you push it through a syringe filter, it can still be pretty viscous (syrupy) and this can clog up the AKTA lines. The AKTA’s really smart - it senses the buildup of pressure and slows down the flow rate - so it starts flowing REALLY SLOWLY. So much for that fast part… So it can be much more time-effective to go with gravity flow in these cases.
Even when it’s not crude lysate, you still need to worry about stuff clogging the tiny tubes - so we filter all of the buffers we send through them - usually using a bottle-top vacuum filter - it has a kind of “bowl” you screw on to the top of the bottle and you pour the buffer into the bowl and it gets sucked through a membrane into the bottle.
Want more details on protein purification and the various forms of chromatography? I have a whole page of posts on my blog: thebumblingbiochemist.com/lets-talk-science/protein-purification/
and a RUclips playlist on protein purification: ruclips.net/p/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7
as well as one specifically on chromatography: ruclips.net/p/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
Are there more advanced videos? This helped a lot when I started but now I am looking at things like which columns to use and in what order, troubleshooting gradient elutions to better purify proteins, choosing different buffers to try and prevent dimerization etc. Is there anything like that on your channel right now? Thx!
I have a whole playlist of purification videos that can hopefully help: ruclips.net/p/PLUWsCDtjESrEtqQkEXsQpTyRfMhywBVQ7