The gravitational force spec consists of an epi mount, a regular light microscope, digital camera, and a computer. We use this to do single molecule acid. We can record force equilibrium or we can provide a grade force by drop.
To start, we need to prepare a glass micro so they can done by these type of process. To measure out about 10 milligrams of the beans that you gonna use, I usually prepare 2K. Here we have five milliliters of we're gonna three triology.
That's where we're gonna get our terminal Put One. Go ahead and mix that up really nice with a transfer and then transfer two milliliters of the mixture on top of your be cake. Leave the beep cakes for two and then vortex thoroughly at two.
The down ate the wet two. You want oh one purine in double distilled water. pH six coupling.
So you simply add five milliliters of the coupling buffer to each beef and this coupling make the bead to ling. So then antibodies the closeup floating ling again, vortex for a minute. Pin them down for five minutes and if the freezer to get your glide, you're gonna cut it down from whatever extent it's to 5%using a coupling buffer.
Then you take two milliliters of this solution add and this will allow the TER couple with the terminal that are already on our vortex. This for two, rotate for three. You coupling buffer procedure.
Again five milliliters, a coupling buffer into your feed cake. Again, mix, spin and aspirate.Sate. On the fourth time some sate on the top of the cake, the antibody to freely move around.
This is our antibody here. Micro that volume. The wet rotate antibodies, you'll seven which is one moly cut with double distilled water and the mixture is rotated under the for 30 minutes for the appropriate time.
To quench all the underact glue, eye to eye. Gonna spin it down, get rid of the S.Then you wanna make a the contents of which are protocol. Take five milliliters of the take the beads, here's the beads, intex down tooth.
Repeat the step three times and then to whatever assay you're doing the same. To start our preparation, we first need to get our protein. In this case go to the negative 70, the keep stored protein, it's important to this protein and heat it up.
This now this right outta the, is gonna be much more concentrated what we wanna use in our assay. So the cut down to about fifteens and gonna put that out some into the tip. But then that has to be because which accurate volume after you out the volume amount down to about five micro this in the large beads, we're gonna take bigger volume, gonna be 10 microliters from this example bead.
And that's gonna give a nice, here we're adding about three 50 more microliters assay buffer into the microcenter tube to give us a final concentration. So now we're going to the do it vi like with aex your will A here we're rotate the one literally and we'll do that. Now it's time to actually construct the flow on which our fees will reside and be.
So I'm putting this standard microscope slide into oh 1%and that be cut with a glue for the anchorings to attach to the surface of the, the glass cut and cover and microscope slides. I've already edges with the, and I'll apply this thinner piece of glass thicker hyper slide. This chamber will be relatively small.
So here's a small here And it's damaged position's. Another one of those Guys hold Every day. The agent makes me better, lack better.
Here we'll pass down the last two 25-year-old possibly taste before we start adding cracker and the cheese. And you've missed sun. Only a few have taste in just a few.
And here if you look closely you can actually see the bead. The tip, what I do is I start by simply tapping on the area I be and I don't, after I do that full while the rest of be the surface of the slide here, we'll at about 40 into be so that we can put this slide through different orientations and not worry about a purposes can migrate down. Hey you don't need no co screw to sample my bouquet for some of my wine, a back of the line and I might get holy Mount the slide and I use an extra clip to keep things more stable And we can put the objective.
This is a remote control we use to position of the here we're slide and everything's turned on including the camera. We can start monitoring for potential be do this by simply moving monitoring the video screen. The one on the left has a smaller bead and its smaller bead kind of looks like the other smaller beads that are just sitting there on the surface of the lop.
However, the bead pair on the, we has a bead that comes in focus when the off bead surface of the LOP that indicates that that be resides in a different of focus than the other be slide and also pair so that be on the right A shows how pairs might work on a slide. So we put the slide as we go through rotation with different angles that gives us different lengths of, we then use rotation. We see a in of what a feed para might look like using a computer.
We find the both. This case calculate we can drop the provide measure and here drop the F and also dropped. Thanks for watching.
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