How To Use The Nano Antibody Kit
Ready for these fluorescent proteins to “light up” your experiments? *Ba dum tss*
We will talk about what precisely a Nanoantibody (nAb™) kit is and how to use it for your specific experiments.
So let’s get started.
What Is A Nano Antibody Kit?
So nAb™ is short for nano antibody, and what they do is selectively bind fluorescent proteins to whatever you want to bind.
For example, our nAb™ kits are named GFP nAb™, which means it binds to a green fluorescent protein, or we will have another kit called mNeongreen nAb™ which will bind mNeonGreen fused proteins.
So the idea here is that you can tag some of your proteins with these fluorescent proteins, and then these kits will help fish them out of the solution.
So let me show you how this works.
What is In A Nano Antibody Kit?
In this kit, you got three different things:
- buffer components
- your GFP nAb™ component
- and all the plastics and the spin columns that come with it
And usually, these buffer components contain:
- 10X lysis buffer
- 20X binding buffer
- 10X wash buffer
- 5X solution buffer
- and 1X neutralisation buffer.
Then you have your nAb™ product, the nano antibody with an agarose bead joined to it. (You can also have a magnetic version of that one.)
And finally, you have your spin column and all the little centrifuge tubes that go along with it.
Everything is included in the kit except the small centrifuge tubes…
Which you got, right?
Step 1. Vortex the nAb™ mixtures
All right, so the first thing that you're gonna wanna do is you are gonna wanna vortex your nAb™ mixtures and vortex it for a couple of seconds to resuspend your nAb mixture.
To begin the procedure, we must remove the plastic piece from the spin column and put it into a 2-millilitre centrifuge tube.
We then add 25 microliters of nAb mixture to the spin column. However, we need to make sure that we the 20X binding buffer into a 1X binding buffer, which is 24 dilutions, before adding 500 microliters of the binding buffer to the spin column and capping the tube.
Now, we need to spin down the nAb mixture/binding buffer, and we can counterbalance it with a random tube that has similar mass and size.
We spin it at 100Gs for about five to 10 seconds and discard the bottom portion before putting the spin column into a new centrifuge tube.
Once we have the spin column in the new centrifuge tube, we resuspend the nAb™ mixture by vortexing the tube for a couple of seconds.
And once you complete that, you have set up your spin column with the nAb mixture, which is ready for further analysis.
Now, what you want to do is you want to have another centrifuge tube that is labelled binding buffer because we'll do that process again. We're gonna wanna discard the old tube and put that in. And then we're gonna wanna spin down again. So we'll do the same thing again with this tube.
Step 2. Add The nAb™ Mixtures To Your Sample
So now I've spun this down with binding buffer twice. I want to cap the bottom because it will be time to add my sample. Here's my fluorescent protein sample, which I will add here. Let's add 700 microliters of fluorescent protein into the spin column.
We'll want to put it into a machine that inverts it many times. Like, you know, do you have one of those guys? And we want to do that for about 10 minutes to two hours. If it's stable, you could do that at room temperature, but if it's not, you want to do it at four degrees C. Don't forget.
Now I know what you're thinking, 10 minutes to two hours, that's a lot of time in between. How do you know what's what? Well, you determine it experimentally because all proteins are slightly different.
So now this has finished incubating. We're gonna wanna remove the bottom and spin this down. So doing that, you wanna keep the contents of the first spin down in case you lose some samples in here. So make sure you keep this tube for the first spin down. Okay? Okay.
So we spin this guy down again. And we're good. So this is the first spin down. You wanna keep the contents of this tube just in case you lose some of your samples. Your sample might be slightly coloured because you lost some of your samples. It fell through. So that's why you wanna keep the first tube. Or if everything's stuck on well, your sample would probably be more colourless.
All right. Now let's go to the wash steps.
So now we wanna transfer this to a clean two-millimetre fusion tube. And now we wanna add more binding buffer again, another 500 microliters. Let's go. Bam.
And then we're gonna wanna spin that down again.
Step 3. The Washing Step
So, this next step is a wash step. So we're gonna do the wash buffer on this one. Another 500 microliters wash buffer. And let's spin this guy down another time.
I filled up this microcentrifuge tube with five microliters of neutralisation buffer. And I'm gonna do this because I will neutralise my sample when it's done. So the final step after I elute, ...
It's gonna go into this neutralisation buffer. So now we have just finished spinning down the sample, and now we wanna cap the bottom and add elution buffer. So now we're adding 50 microliters of elution buffer in here. Just 50 microliters. We wanna resuspend this guy up and down for 30 seconds.
So now, this is my neutralisation buffer tube. I'm gonna remove this cap and put that into my neutralisation tube. And sometimes, I might have a little sample in the cap, and I wanna make sure I pipette this out and put it back in the tube to spin down. So now, again, let's go spin this guy down for the final time.
Step 4. Elute Your Sample
This is the final step. We have the elution buffer in here and the neutralisation buffer on the bottom, and we're gonna spin it down to elute our sample, which is the nav plus the GFP complex, into the neutralisation buffer. And then you're gonna be done. 30 to 60 seconds at a thousand Gs. So if you want higher recovery, you want to repeat the elution step one more time. That means you add the elution buffer again, pipette up and down for 30 seconds and then spin down the sample into the neutralisation buffer again.
So now that your sample is eluted, you can do what you want with the sample. You can analyse it by spectrophotometry, fluorometry, or whatever you need to do with the sample. And that's how you use the kit.
So, that’s how you use the nano antibody kit for your research. Suppose you have any questions on the exact process. In that case, you can always leave a comment on the YouTube video below, or if you have purchased one of the nano antibody kits from us, you can always click here to go to our support page to contact us via email or live chat. Have a wonderful day!