00:01
The last technique I wanna talk
about here is that a proteomics.
00:05
So far we have talked about how to
replicate DNA in incredible quantities.
00:08
We have talked about how to analyze the
transcriptome or all the messenger RNAs of a cell.
00:15
Now we are gonna apply our technologies to
understanding all of the proteins in a cell.
00:22
So in proteomics that's what we are after.
We want to accomplish the same end
proteomics that we had in transcriptomics.
00:30
We have to use some different techniques to accomplish
that; because, of course proteins are different from RNAs.
00:37
One of the techniques that we used to do this
is called 2D gel electrophoresis to
accomplish it. So let me explain how that works.
00:45
There is two primary step process in this.
00:48
First, one takes a protein mixture
that one has gotten from cells
and applies it to what's called a polyelectrolyte
column. Now this is a column that has
material in it that has a variety of charges on it.
01:01
And charges, as you know, will segregate
themselves according to the
electrical field that you put them into.
So if I have a tube of this material
and I apply an electrical charge across it.
01:13
And let's say I put a positive at the
top and a negative at the bottom
then all of the ions that are in that column
the negative ones that are the most negative are
gonna travel the furthest to the top, close to the positive.
01:25
And the ones that are positive are gonna travel
furthest down to the bottom closes to the negative,
and between the two we will see a gradient. In the middle
there be things that will have very little charge
and then growing out to very large charges later.
01:41
Well what I have just done is that I have created
a gradient of charge and that gradient of charge
is very powerful for allowing me to separate
proteins who have a variety of charges,
okay? The proteins will separate according
to their pI values, the place
where the charge is 0. They will go to the
place where charge is 0 and they will stop.
02:02
As so this gradient is a really powerful
technique, the ones that are most positive
and neutral pH are gonna be down below and the
ones that are most negative are gonna be up at the top.
02:13
I'll take that gel. Now, this gel has already separated
these proteins according to, basically, their charge.
02:19
It has separated them according to their charge and
I lay this gel on top of another gel.
02:26
So, I have just done what we
called the first dimension of the 2D gel,
separated the proteins on
the basis of their charge.
02:35
This column that I have just
created is now applied
to another gel that is going to separate
them on the basis of their size.
02:44
So to do this I add a detergent called SDS.
02:48
And what SDS does is, it kinda neutralizes
all the charges. So they don't effect
the next analysis.
02:54
The next analysis is going to separate them
only on the basis of their size not on their charge.
03:01
So we apply an electrical current from the top to
the bottom and all of the proteins moved into the gel.
03:07
And the difference in this gel is the
speed with which they move through it
is a function of their size.
03:15
The smallest proteins will move the
fastest. They travel the easiest route.
03:20
The largest proteins will be
those that travel the slowest.
03:23
They have a more difficult time getting
through the pores of the gel.
03:29
So the 2D are charged or pI
have a role to think about that
and the second dimension moving down in terms of size.
03:36
Well this is a result that you get
if you do that kind of an analysis.
03:40
You see all the proteins of a cell
now spread out into a 2D pattern.
03:46
The most positives and the largest,
in this case, on the left.
03:52
The most negative and the
largest on the right.
03:54
The neutral in the center and then by going up
and down we can have the biggest and the smallest.
04:01
So for example I might know that a protein
that I am interested in studying has a certain size.
04:05
It has a certain charge and I could find on this
graph the spot of that protein corresponded to.
04:13
The beauty of this technique
is, I can analyze
every single protein; because, every spot
on that gel corresponds to one protein.
04:22
And if I make my gel big enough I can get a spot
for every single protein that's found in a cell.
04:29
Well, if you think about what we did in
the last analysis where we compared
the healthy cells versus the cancer cell,
you can get an idea about what we
can do with 2-D gel electrophoresis;
because,
let's think about that, if we start
about with 2 sets of cells
a healthy one and a cancerous one,
then what we do is we label the
proteins in one of them orange.
04:53
And we label the proteins in the
other one, the cancer cell, blue.
04:57
When we run those on the
gel, this is what we get.
05:02
So now we can see some differences corresponding to color
like what we saw in the
transcriptomics analysis.
05:09
Here are orange that are proteins that are
in a healthy cell, but not in a cancer cell.
05:13
Things that are purely blue are proteins that
are in a cancer cell but not in a healthy cell.
05:19
And proteins that are black are proteins that
are equally abundant in both cells.
05:25
Now this kind of information can compliment
the transcriptomics analysis very nicely to
help us give a very precise piece of information
about what cells have in them
and then compare them between
two different types of cells.
05:39
And this doesn't have to be a cancer cell.
We could do the same kind of an analysis
for either transcriptomics or for
proteomics. Well let's say a drug.
05:47
May be we are interested in understanding the
effect that a drug has on a healthy cell.
05:52
Where we take a health cell without a drug, take the
same healthy cell with a drug and perform this analysis.
05:59
In many ways researches are
limited only by their imagination
in terms of the kinds of information
they can ultimately get out.
06:06
Well, we have learned about three different
techniques for analyzing the information in cells.
06:11
The information being the DNA, the RNA and the protein.
06:16
I hope that you have come away with
the understanding that these techniques
allows to determine things at a system level that
would otherwise have been impossible 30 years ago.