• AMA with Certified Student Loan Professional

    Join SDN on December 7th at 6:00 PM Eastern as we host Andrew Paulson of StudentLoanAdvice.com for an AMA webinar. He'll be answering your questions about how to best manage your student loans. Register now!

AAMC FL2 Question for Biological Sciences (HELP!!)

Silent_C

New Member
Aug 4, 2017
8
2
  1. Pre-Medical
    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!

    Screen Shot 2017-08-15 at 6.42.21 PM.png
     

    Nugester

    Full Member
    2+ Year Member
    Jul 4, 2017
    842
    824
    1. Medical Student
      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.
       
      Last edited:

      Silent_C

      New Member
      Aug 4, 2017
      8
      2
      1. Pre-Medical
        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?
         
        About the Ads

        workaholic181

        Full Member
        2+ Year Member
        May 29, 2017
        1,292
        850
        1. Pre-Health (Field Undecided)
          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

          Full Member
          2+ Year Member
          Jul 4, 2017
          842
          824
          1. Medical Student
            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

            Full Member
            2+ Year Member
            Jul 4, 2017
            842
            824
            1. Medical Student
              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

              Full Member
              5+ Year Member
              Nov 1, 2015
              5,300
              4,065
              1. Medical Student
                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?
                 
                This thread is more than 4 years old.

                Your message may be considered spam for the following reasons:

                1. Your new thread title is very short, and likely is unhelpful.
                2. Your reply is very short and likely does not add anything to the thread.
                3. Your reply is very long and likely does not add anything to the thread.
                4. It is very likely that it does not need any further discussion and thus bumping it serves no purpose.
                5. Your message is mostly quotes or spoilers.
                6. Your reply has occurred very quickly after a previous reply and likely does not add anything to the thread.
                7. This thread is locked.