How to clean and prepare a super loop for use in an AKTA FPLC system
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- Опубликовано: 24 авг 2024
- This is a video tutorial on to clean the super loop/superloop for FPLC protein separation. It shows you how to disassemble, clean, reassemble the loop. Then, we connect it to the AKTA purifier and wash it with buffer A.
Thank you for this demonstration, exactly what I was looking for :)
Thanks for this!
Please share more videos on Akta sample loading using Pump p950
Hi Hari! That's actually something I don't have a lot of expertise on. My postdoc has used the sample pump in one of his former labs, but it was always one of the luxuries that we never purchased in the labs I was trained in. As a result, I built up some mighty strong forearms during my postdoc years!
What's the process for putting protein sample into the loop next?
The standard way is to load a disposable syringe with your sample (anywhere from 1-50 mL), attach a 0.45 um syringe filter to the end, dispense enough of your sample so that it fills the syringe filter and that there are no bubbles at the tip, and then simply inject your sample into the sample port in the injection valve. The default state for the injection valve, if no program is running in Unicorn, is the "load" state, which means sample injected into the load port will go into the loop as you inject. I never take this for granted, and I'm always looking at the super loop as I inject to make sure the stopper is moving up as I add my sample. I also look at the waste line from the injection valve: as I inject sample into the loop, buffer is displaced from the top of the loop, and that buffer gets directed to waste.
Once my sample is injected, I can either start a program or manually run the sample. Most of my trainees do nickel and ion exchange columns manually these days, and we only use programs for our gel filtration columns, but either way, once the sample is in the loop, you can do what like from there.
A final note: sometimes using the 0.45 um syringe filter makes it really hard to inject your sample: You're ultimately trying to shoot typically 25-50 mL of viscous solution through a tiny channel carved in the injection valve (not to mention the PEEK tubing). Even without the filter, injecting it carefully without breaking the luer lock fitting can require a lot of upper body strength and control. I always tell my students to take their time and take a break if their arms get tired. It's not a race. However, if the syringe filter is making things too difficult, a hard spin in the centrifuge at 20-40,000 x g for 30 minutes is often enough to get most of the large insoluble material out of your sample, and if you do this step you can usually eliminate the filter.
Hope that helps!
@@FitzkeeLab that was very helpful, thank you!!