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- Welcome to the first video
in our Experiment Explained series.
The goal of this video series
is to provide you with a short, digestible
and high yield reviews of experimental techniques
that you're likely to see on the MCAT.
My name is Vikram Shaw
and today we'll be looking at everything you need to know
about western blotting.
We'll look at figures, controls
and common places you'll see western blots in the biology
and biochemistry section of MCAT.
By the end of this video,
you'll feel confident in your ability
to analyze western blot experiments
that may show up in any context on MCAT.
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The western blot is widespread technique
that researchers use to measure
the amount of protein in a sample.
Don't worry if that doesn't make sense to you quite yet,
we're gonna take a step back
and walk you through all of the basic science
and logic you'll need to know to understand western blots.
To start, let's go all the way back
to the central dogma of biology.
Fundamentally, the central dogma describes the process
by which the instructions present in our DNA
are converted into products
that our cells can use to carry out their functions.
While recent discoveries have uncovered some additions
and exceptions to that rule,
this simple schema is what you'll need to know for the MCAT.
The DNA inside of ourselves
is transcribed into RNA in the nucleus.
That RNA is then exported to the cytoplasm
and undergoes some forms of processing.
Cellular ribosomes, either free-floating
or associated with the rough endoplasmic reticulum,
then convert the RNA instructions
into a functional protein product.
When it comes to the western blot,
this protein product is what we're measuring.
Here's an example of what a western blot looks like.
We won't cover the specific steps
for how to process the sample
and conduct the gel electrophoresis in this video.
Instead, we're gonna focus on how the MCAT
will ask you to identify
the results of a western blot quickly and accurately.
So let's break down the anatomy of this western blot.
First, like every good experiment,
western blots have an independent variable or variables,
a dependent variable or variables and in control.
In this western blot,
there are several different independent variables.
At the top of this figure are various siRNA molecules,
such as a control or different IRE1s.
The researchers are also measuring each siRNA condition
in the presence of either a mock or a salmonella infection.
These are all independent variables.
The number of combinations of independent variables
can quickly become complicated,
which is why a western blot is a convenient way
to depict a large amount of information.
For example, the western blot gives us a convenient way
of measuring the protein expression
of the mock infection in the IRE1 number one condition
to the protein expression of the salmonella infection
in the IRE1 number three condition.
Let's now look at the dependent variable.
In this case, researchers are interested
in the expression of the E2F1 protein.
This information can actually be inferred from the figure
and without any additional information,
though, the passage that accompanies this figure
will likely have provided helpful context.
E2F1 must be the dependent variable because beta-actin,
which stays relatively constant
given the same band size we see across the bottom,
is a commonly used control.
Further, E2F1 levels vary depending on the condition.
While some researchers use western blot
to quantitatively measure information,
it's generally used as a qualitative tool.
We can visually see without needing numbers
or to count individual pixels
that the protein expression level is lower
for the control salmonella condition
compared to the other conditions.
Let's talk a little bit more about the controls
you might see on a western blot.
Researchers often use proteins such as beta-actin, tubulin,
or other highly expressed cellular proteins
to ensure that the same amount of sample
is loaded in each lane of the gel.
These are proteins that are well-known
and behave predictably.
In other words, to confirm that our differences
in E2F1 are due to the independent variable,
the experimenters can control for human error
and ensure that the same amount of protein
was loaded in each lane of the gel
by including that beta-actin control.
Finally, on the right side of the gel,
notice the numbers that say kDa.
This stands for kilodaltons
and it's a unit for measuring the weight of the protein.
The lighter of the protein,
the further down it will appear on the resulting gel
and therefore the smaller kilodalton weight it'll have.
Now that you have a good grasp of western blot anatomy,
let's look at a couple of questions
you might see on the MCAT.
Before looking at the question,
let's take a couple of seconds to analyze
what's going on in this western blot.
Starting at the top,
there are a few different independent variables.
For example, either 0.5 millimolar of calcium alone
or one millimolar of calcium was used
with one micromolar of the R568 compound.
Pi, expressed in millimolar
and the pH are also independent variables.
The dependent variable
is protein expression levels of phosphorylated ERK
and the control is beta-actin.
That was pretty easy, right?
Now, let's look at the question.
The question asks, the researchers include beta-actin levels
in figure one in order to, A, controlled for cell type,
B, control for pH levels,
C, control for the amount of protein loaded in the gel,
or D, control for unwanted proteins.
Recall that controls are used to ensure that researchers
load the same amount of protein in the gel,
which in turn helps the researchers
ensure that differences in their dependent variable
are indeed true differences
and aren't just due to experiment or random error.
Therefore, the correct answer here is C.
Let's look at another western blotting question.
Now, before we look at a question,
I'd like you to work on a quick exercise.
Go ahead and take a moment
to make a list of the independent variables,
the dependent variable
and any controls you see in this western blot
now that we've done it a couple of times.
Once you're done, unpause this video
and let's check your answers.
So it looks like there two independent variables,
one, the sample is either from the eye or the scalp
and two, the sample is wild-type
or contains a serine to leucine mutation
at the 743 amino acid position.
The dependent variable is protein expression of OCA2
and the control is, once again, beta-actin.
Let's take the analysis one step further.
Are there any patterns that we see?
Looking at the western blot,
it appears that in the S743L condition,
ACA2 protein levels are lower than wild-type ACA2 levels
in both the eye and scalp samples.
In addition, both of the scalp samples
have lower ACA2 protein expression
compared to the eye samples.
Although this extra analysis
might take a little bit of time,
it might be useful to obtain this information
before moving on to any questions during the MCAT,
especially if you can do so quickly.
Now, if you're wondering what the graph to the right is,
that's actually a quantification
of the western blot found to the left.
Although this is a bit of an oversimplification,
the quantification is generally calculated
by using computer software to count the pixels.
The quantification is helpful because it shows us
if a difference in protein expression levels
is statistically significant.
Here, the asterisks indicate
that there is a significant difference
between protein levels within the wild-type
and S743L conditions in both the eye and scalp samples.
Let's now move on to our final question.
Based on figure one, which of the following conclusions
is most likely to be correct?
Now, before I read the answer choices,
remember that we concluded
that there's a statistically significant
lower ACA2 protein expression in the S743L mutant condition
in both the eye and scalp samples
when compared to wild-type.
I know that's a mouthful, but let's keep that in mind
as we go through our answer choices.
Answer choice A reads, the S743L mutation
decreases ACA2 transcription.
That looks like a great answer,
but there's one key piece that's incorrect.
Remember, a western blot measures protein levels,
which is a result of translation.
Answer choice A focuses on the transcription of DNA to RNA.
Information about transcription
can't be provided with the western blot.
Similarly, when we read the answer choice B,
wild-type mice contain higher levels of ACA2 transcripts,
we can quickly eliminate the answer
since it's talking about RNA levels and not protein levels.
Choice C reads, S743L have lower levels of ACA2 protein.
This actually aligns nicely with the conclusion
that we see statistically significant
lower ACA2 protein expression in the S743L mutant condition
in both the eye and scalp samples
when compared to wild-type.
The answer choice confirms the prediction we had made
after analyzing that western blot.
So this answer choice has a good candidate
for the correct answer option,
but let's look at our final answer choice.
Choice D reads, wild-type mice
translationally upregulate ACA2 protein.
While it's true that wild-type had higher ACA2 levels
compared to the S743L mutant condition,
we can't conclusively say that this is due to the process
of translational upregulation.
Wild-type mice could have had higher ACA2 levels
for many other reasons
that aren't strictly translational regulation.
For example, what if wild-type mice
transcriptionally upregulate ACA2 mRNA
so that we see more protein expression in those samples.
If you can think of a counter example to any answer choice
on any section of the MCAT,
there's a pretty good chance that that answer is incorrect.
After looking through all four of our answer choice options,
we can say that answer choice C is our correct answer here.
Finally, I'll leave you with one more piece of information
you'll find helpful in your study.
The MCAT may test you about other blotting techniques
such as northern and southern blots.
southern blots measure DNA, Northern blots measure RNA
and western blots, as we discussed today, measure protein.
You can use the easy mnemonic, SNOWDROP, to memorize this.
The S and D correspond to southern blots and DNA,
the N and R correspond to Northern blots and RNA
and the W and P correspond to a western blots and protein.
The western blot and related experimental techniques
often appear in the biology
and biochem section of your MCAT.
Throughout this video,
we've learned what a western blot looks like,
we've learned the anatomy of a western blot
and we've learned how to apply your knowledge
of western blots to practice questions.
We hope this video has been a helpful resource
for you in your MCAT study.
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