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ELIZABETH NOLAN: Today's recitation

will all stem from this reading by Youngman and Green

on ribosome purification.

But also, we posted an optional-- well,

not really optional but an additional reading

that's a review that talks about purifying macromolecular

machines from the source.

And I guess one thing I'd just like

to say about this review, something

I like is figure three because it indicates

many, many different types of methods that can be used

to purify some biomolecule.

And I think often it's easy just to think all the time,

well, let's just use a tag.

And tags have enabled many things,

but there's also many possibilities

out there and decades worth of work

before tags for how to purify proteins.

And in some instances, you might not want to use a tag

and that's discussed to some degree here.

So I guess I'm curious.

What did you all think about this week's paper?

Did you like it?

Did you not like it?

Was it easy to hard read?

Did you read the paper?

AUDIENCE: It wasn't too bad.

I've purified--

JOANNE STUBBE: You need to speak louder

because I can't hear you.

AUDIENCE: It wasn't too bad.

I've purified proteins before, so I

felt like I could follow along.

ELIZABETH NOLAN: So not too bad means

it was easy to understand and follow the text there.

Yeah, so compared to the reading for recitations two and three,

this one was probably easier to work with,

but there's a lot of details in this one too there.

One thing, I guess, I like this paper from the standpoint of it

being a methods paper, is the amount of detail

they give with how they did this purification and things that

didn't work, right?

So often, sharing those details with your readers

can make such difference for an experimentalist

in another lab in another part of the world, right?

And I think, from this, one with biochemistry training

could go into the lab and reproduce their purification

there.

So it's a good example in terms of what details to include

and what types of pitfalls to include.

So how many of you have done a protein purification,

either in a lab class or in your research?

OK.

And so what type of purification did you use?

AUDIENCE: We used a histamine-tag with nickel.

ELIZABETH NOLAN: OK.

So you used a His6-tag, probably a polyhistamine tag

in a nickel NTA column.

For everyone else, has it been that type of methodology

or another methodology?

AUDIENCE: I used the same one.

ELIZABETH NOLAN: OK.

OK.

So would someone like to kind of comment

on the basics of affinity tag purification?

So how does this work?

And why did you do it?

So what are the advantages?

AUDIENCE: So you have a light chain

of histamines on your protein, and you

use nickel, which is a metal that chelates

to histamine very well.

Is that right?

ELIZABETH NOLAN: Yeah.

The nickel's bound to something though.

You have the NTA ligand on the resin.

AUDIENCE: I did this over a year ago.

Sorry.

AUDIENCE: So if you have whatever

the type is, the [INAUDIBLE] bound to some solid substrate.

And then you can elute everything

that's not bond to that.

Elute what it does bind.

So you just tag the protein.

It's bound to a high concentration

of the free ligand.

ELIZABETH NOLAN: Or right, to push it off.

So the idea is that you have your biomolecule of interest,

whether that be a protein, which is what we're all

most familiar with, or the ribosome,

and then you attach a tag.

And that tag can be any number of things, right?

So in this paper, we saw they used a stem loop structure

incorporated into the 23s rRNA.

And the idea is that you're going

to use that tag to separate your biomolecule of interest

from the complexity of the cellular environment

there, so all of the other proteins.

And so you have some bead or resin

that this tag can bind to.

So in the case of the nickel column,

the His6-tag will bind to the nickel NTA on the resin.

And then you can wash away other components.

And then you devise some method to elute the protein

you hope to have trapped there.

So what are some advantages of using an affinity tag,

just thinking about this generally

before we delve into the paper?

AUDIENCE: Easy to install.

ELIZABETH NOLAN: OK.

So what do you mean by easy to install?

AUDIENCE: If you wanted to just encode

a 6 His-tag at the terminus of your target protein,

I don't know if you can say it's trivial.

It's easy.

ELIZABETH NOLAN: I'd agree it's easy.

There's many plasmids available that you

can insert your gene of interest into in order to have

this tag genetically encoded.

So when you express the protein, the tag's there.

So beyond that, from the standpoint of purification--

AUDIENCE: It's more specific.

ELIZABETH NOLAN: Pardon?

AUDIENCE: It's more specific.

ELIZABETH NOLAN: More specific--

AUDIENCE: Pure proteins as opposed to various charges.

AUDIENCE: It simplifies the purification.

Instead of doing size exclusion and ion exchange

that is much different.

ELIZABETH NOLAN: So the hope is it simplifies the purification

because you have some way initially

to pull your protein of interest out of your cell lysate there.

So that can be a big help.

What are some potential disadvantages of using a tag?

So have any of you run into trouble with a tag in the lab?

AUDIENCE: Having a tag can highly deform your protein

and change it.

ELIZABETH NOLAN: Yeah.

So it might change your protein or deform it.

What do you mean by "deform?"

AUDIENCE: It could just cause a conformation change

or the tag could make it localize somewhere else based

on size.

ELIZABETH NOLAN: Yeah.

So that's an example, say if you were doing a study in cells,

say, rather than a purification.

But maybe you tag a protein and it

goes somewhere other than it would go untagged right?

And that will affect your observations

and your data there.

And it might alter the conformation.

So it might affect the folding, right?

It might affect the oligomerization.

His-tags bind metal ions.

So is that a factor to consider there?

If you have an enzyme, will the tag affect activity, right?

And these things can be a positive or a negative.

Sometimes the tag is helpful in these regards.

You can't get soluble protein without the tag, right?

And sometimes the reverse.

You decide to express your protein or biomolecule

with a tag and you find out you get an aggregate, something

that the protein shouldn't be.

So these are just things to keep in mind when designing a fusion

protein and thinking about how you're

going to use an affinity tag to express your protein there

and purify your protein.

So there's pluses and minuses, right?

And you can always make the choice not to use a tag.

So you saw some of that in the review article, right?

And I guess one other thing, too, there's

this idea of using the affinity tag in the affinity column,

which we'll talk about more in the context of the ribosome,

but is that always enough?

So is the affinity column alone always enough

to purify your protein of interest?

So in lab class, that's where it will end,

because that's an exercise made for lab class,

and it's your first adventure into protein purification

for most people, right?

Oftentimes, it's not enough.

That you do enrich what you've purified with what you want,

but often there's contaminants.

So you actually do move forward with doing some other type

of purification.

Like Rebecca mentioned, ion exchange or size exclusion,

those are things you can use after the affinity purification

there as needed, right?

So contaminations are something to look out for here.

JOANNE STUBBE: What other types of steps

do you use for purification besides columns

and [INAUDIBLE].

Because people have forgotten all of this.

Everybody uses the tag and that's the end of it,

and it can be the kiss of death.

What other kind of fractionation steps?

Do you do any other fractionation steps?

What is it, 535 or something?

AUDIENCE: [INAUDIBLE] salt.

JOANNE STUBBE: The what?

AUDIENCE: The [INAUDIBLE] salt. So

some proteins will precipitate.

Some will not.

JOANNE STUBBE: Yeah.

So that's what you use to precipitate.

So that's a mild method.

It's fast.

It gives you separation on a fair amount of proteins.

Anybody know what you use?

AUDIENCE: Ammonium sulfate.

JOANNE STUBBE: Yeah, ammonium sulfate.

And then what's the other thing that really is important?

What is the other thing you want to remove from your protein

when you're using this tag that oftentimes people miss

in the literature?

ELIZABETH NOLAN: Yeah, we were getting there.

JOANNE STUBBE: There's another component inside the cell

that you need to get rid of that you've been talking about.

AUDIENCE: Where one component has His-tag on the cell,

you remove it.

JOANNE STUBBE: So you have the protein.

What are the other components in the cell

that you need to remove?

You can go back and look at your cartoon

of the inside of the cell.

ELIZABETH NOLAN: Yeah.

So we're getting a little ahead, but that's totally fine.

So if you're going to lyse your cell, and then,

imagine your protein's soluble, right?

So you do a centrifugation to remove

the insoluble components.

So you have the membrane and all that debris.

And then you take your soluble fraction,

and you incubate that on your column, right?

And then you elute, you wash, you elute your protein.

What might come out with your protein?

AUDIENCE: Nucleic acid.

ELIZABETH NOLAN: Yeah, right.

So how do you know if you have nucleic acid contaminating?

AUDIENCE: The A260.

ELIZABETH NOLAN: Right.

So A260 will give you a readout of nucleic acids.

A280 is what people typically look at for their protein

concentration, but you should look at both so you

know what's in your protein.

You need to look at both.

JOANNE STUBBE: Do that and adaptively

repeat stuff out of the literature.

Very frequently, you take an absorption spectrum,