AAMC FL2 Question for Biological Sciences (HELP!!)

[Silent_C]

Hey guys!
I'm currently stuck on this question that I posted below.
How can you analyze the "rate" of histone acetylation by western blot?
From my knowledge, I thought Western blot just shows you whether the protein is there or not and the relative amounts (compared to control).

Why couldn't RT-PCR detect levels of expression? The increase in HA means an increase in gene expression, which RT-PCR analyzes as well.


Thank you!

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[Nugester]

Since I have done a lot of western blots in the past, yes, you can quantify western blots. Your protein of interest (deleted gene) is compared to the normal gene product (normal protein). You compare the signal intensities via software. Assuming deletion in gene = less protein, the signal would appear weaker compared to the normal protein. To make sure this isn't because the researcher didn't load different amounts of protein to get this result, you have a control protein like Actin, which is the same with the deleted gene and normal gene.

I believe you are confused because they mentioned rates. With westerns, you can set up experiments to measure protein levels after 1 day, 2 days, 3 days etc or even minutes!

You could also answer this by POE.

B. Southern - DNA
C. Northern - RNA
D. RT-PCR - RNA->DNA

A is the only thing "different" so best answer is A.

 

[Silent_C]

Since I have done a lot of western blots in the past, yes, you can quantify western blots. Your protein of interest (deleted gene) is compared to the normal gene product (normal protein). You compare the signal intensities via software. Assuming deletion in gene = less protein, the signal would appear weaker compared to the normal protein. To make sure this isn't because the researcher didn't load different amounts of protein to get this result, you have a control protein like Actin, which is the same with the deleted gene and normal gene.

I believe you are confused because they mentioned rates. With westerns, you can set up experiments to measure protein levels after 1 day, 2 days, 3 days etc or even minutes!

You could also answer this by POE.

B. Southern - DNA
C. Northern - RNA
D. RT-PCR - RNA->DNA

A is the only thing "different" so best answer is A.

Thank you for the reply! I was confused about the rates, yes, but also because RT-PCR can detect levels of expression because its looking at mRNA (which is RT to cDNA in order to quantify). So why can't we use RT-PCR?

If there is more histone acetylation, that would mean increased gene expression which can be detected via mRNA/cDNA quantification, no?

 

[Silent_C]

Sorry one more question! When do you decide when to use RT-PCR vs. Northern blot?

 

[workaholic181]

Thank you for the reply! I was confused about the rates, yes, but also because RT-PCR can detect levels of expression because its looking at mRNA (which is RT to cDNA in order to quantify). So why can't we use RT-PCR?

If there is more histone acetylation, that would mean increased gene expression which can be detected via mRNA/cDNA quantification, no?

Because the question asks for the BEST experimental method. Western blots is the most straightforward way to test for proteins, thus it is the best technique.

Just know what each test looks for and you'll be fine.

 

[Nugester]

Thank you for the reply! I was confused about the rates, yes, but also because RT-PCR can detect levels of expression because its looking at mRNA (which is RT to cDNA in order to quantify). So why can't we use RT-PCR?

If there is more histone acetylation, that would mean increased gene expression which can be detected via mRNA/cDNA quantification, no?

Because they are interested at the protein level, not mRNA level.

 

[Nugester]

Sorry one more question! When do you decide when to use RT-PCR vs. Northern blot?

You can get an absolute quantification with RT-PCR, while it is relative with northern. RT-PCR is much faster and there are many things that can go wrong when using northern blots. For the question, I would focus more on what each method detects; as I mentioned before 3 of them deal with RNA/DNA, so westerns the best choice.

 

[aldol16]

How can you analyze the "rate" of histone acetylation by western blot?

Do quantitative Western blots at different time points after gene deletion to get the relative rates.

Why couldn't RT-PCR detect levels of expression? The increase in HA means an increase in gene expression, which RT-PCR analyzes as well.

RT-PCR does detect protein expression. But how would it detect post-translational modifications, which is what is happening here?