Name: 7. Kevin Ahern的《生物化學--蛋白質純化II》。 (7. Kevin Ahern's Biochemistry - Protein Purification II)
Uploaded: 2021-01-14T05:20:28.000Z
Duration: 46 min 36 s
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Monday, Monday, I have a song I'm writing

called Hyundai, Hyundai, I'm sure you can figure out

We spent some time last time talking about techniques

and today I'm going to spend some additional time

some of which I think you'll find very medically relevant.

So, hopefully, you'll find this interesting what I have to say.

talking about polyacrylamide gel electrophoresis

and more specifically, I talked about SDS-PAGE and SDS-PAGE

is what we use to separate proteins by electrophoresis

to make them behave like DNA molecules so we manipulate them

so they behave like DNA molecules and the manipulation

that we did was that we treated them with SDS.

The SDS coated those proteins and it made them lay out flat

like rods and the coating gave them a very negative charge

The common question I get, and I got last time was,

"Well does that negative charge negate any other charge

And the answer is yes it does because there's a lot more of those SDS's

on there than there are charged residues within that protein.

So that effectively makes that a polyanionic substance

meaning there are multiple negative charges.

Some proteins behave a little anomalously but the vast majority

The next figure I want to show you is an example of a protein gel

where on both sides of this is called a ladder

and a ladder simply is a set of proteins of known size

that are there and then you can see a protein being purified

at various steps in the purification process

and there are various quantities of that protein

being present and we can see an estimated size from that.

As I noted last time, this technique really isn't useful

for isolating a protein that we want to use later,

it's more of an analytical technique that tells us

how much protein do I have here, what's its size

and how pure is it compared to the things

That's what SDS-PAGE is very, very useful for.

SDS-PAGE as we shall see is very useful when combined

with another technique that I'm just getting ready

to talk to you about and so I'll do that in just a moment

but before I do that I'll remind you of the structure of SDS

and no you don't have to draw SDS but this is what it looks like.

It's a long chain fatty acid and these are all carbons

and hydrogens down here and at the end we've got a sulfate

the negative charge that is necessary for electrophoresis.

Well, as I said, if we combine SDS-PAGE with another technique,

we get a really, really powerful technique

and it's a powerful technique for studying things like cancer,

for studying how drugs work and so forth, so I want to spend

a few minutes talking about that because I think

it's a pretty cool and fascinating technique.

The technique I want to discuss first is called isoelectric focusing.

Isoelectric focusing and this technique uses some substances

we don't have to talk really too much about them

but suffice it to say that it relies on a mixture of compounds

Some of them are very, very, very negative

and some of them are very, very, very positive

and some of them are in between, so we can imagine,

for example, that we might have a compound that might

have forty negative charges and another compound

in our mixture that might have thirty-nine, thirty-eight,

thirty-seven, thirty-six negative charges and on the other side,

we might have something that has forty positive charges

and thirty-nine, thirty-eight, thirty-seven.

And so, we've got a complete spectrum of charges

Well if I take this mixture and I treat it very carefully,

and I put it into a tube kind of like the columns

that we've been talking about and apply an electrical field

to that tube what will happen is that the things

that are the most negatively charged will move toward

And if I look at that tube when I've finished

doing this electrical treatment of the tube

what I'll see is that the things that had forty minus

will be closest to the end followed by the thirty-nine minus

the thirty-seven minus, etcetera, etcetera.

And in the very middle that's where I'll see things

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7. Kevin Ahern的《生物化學--蛋白質純化II》。 (7. Kevin Ahern's Biochemistry - Protein Purification II)

material

technique

negative

determine