Desalting columns for desalting and buffer exchange скачать в хорошем качестве

Desalting columns for desalting and buffer exchange

I’m a desalting column, short and stout - small things stay in while bigger things flow out. The reason they do this is that they take a shorter route, so put your sample in and exchange the buffers out! Let’s take a look starting a way you probably won’t find in a book… blog: https://bit.ly/desalt_buffer_exchange Imagination you have a turtle race with a crowd of turtles en route and then partway through the race a cheetah and a crowd of tortoises come in. The cheetah’s gonna beat all those tortoises, but since the turtles had such a head start, it’ll finish in a crowd of them. And if you stop the race before the tortoises come in you get that cheetah in a crowd of turtles (the starting racers) instead of in a crowd of tortoises (the partners it came in with). Now imagine that, instead of a cheetah, you have one of those giant tortoises which are so awesome. They don’t move faster than the turtles, but they get to take a shortcut. They’ll still finish ahead of the turtles they come in with that don’t get to take the shortcut, but they’ll finish with the starting crowd. This is the concept behind buffer exchange You have a column filled with resin (little beads) and those bead have “secret tunnels” - winding pores that only molecules small enough to enter them can go through. Molecules (be they proteins, DNA, RNA, etc.) that can’t fit get to go around (shortcut!) but molecules that can fit (like salts, free ATP, etc.) have to go the long way. And because they have to travel further, they take longer to go through. Before I talked about how I use size exclusion chromatography (SEC) during protein purification as a “polishing step” to separate the protein I want from proteins that are different sizes. In that case, I want good resolution (ability to separate similar-sized things) - so I want a “long racecourse” so that the molecules encounter more beads, with smaller things getting slowed down more and more and you can catch the outgoing molecules on their way out by taking fractions. If you want good resolution, you want a long, skinny column with little beads But if you want more of a yes/no resolution (like salt vs protein) you want a short, fat column and big beads. G-25 is the name of the resin, which is a form of “Superdex” - the dex is for dextran - a sugar that, along with agarose, forms the beads’ gel mesh. These versions are small, perfect for the small volumes I’m using, but they also have bigger ones, including ones you can use for proteins. In undergrad, I used PD-10 columns for buffer exchange of proteins - those are smaller volumes of the HiPrep desalting column I’m currently using. The DESALTING part comes in handy for things like ion exchange chromatography (IEX), where you get a protein to bind to a column based opposite-charge attractions, then you add increasing levels of salt to outcompete it (when a salt dissolves it breaks into its component ions (charged particles) - e.g. table salt (sodium chloride, NaCl) becomes Na⁺ + Cl⁻. This can leave you with a very salty (though hopefully also very pure) protein. To remove that excess salt you could use dialysis, where you put the protein in a semipermeable-membraned pouch and put it in a bunch of low salt buffer - protein stays in the pouch & salt flows out until concentrations of salt equalize inside & out. Cheap & does the trick, but takes a while… you can instead use a desalting column which you “pre-fill” with the liquid that you want your protein to come out with. Also good for removing small competitors like imidazole that you might use for affinity chromatography. more on other size exclusion chromatography resins: http://bit.ly/sizeexclusionchromatogr... more on dialysis: https://bit.ly/proteindialysis more on protein chromatography: http://bit.ly/proteincleaning more on radiolabeling: http://bit.ly/radiolabelings more on oligo synthesis: http://bit.ly/2We8e8W           more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com                                          #scicomm #biochemistry #molecularbiology #biology #sciencelife #science #realtimechem