Expert: Paras Anand - 5/9/2009

Question
Hi, thank you so much for taking the time to look at my
question.  I am a third year Biology undergraduate student
and am trying to get some ideas of how to design good
experiments.
What is the best way of testing where a protein is
expressed? I know you could tag the protein, but then how
would you find out where it was expressed without having to
extract tissue from every cell in the body??! I have also
heard of expression studies where a promoter for a gene of
interest is taken from another cell and then inserted into
the new cell where the gene of interest has been replaced
with GFP. So the GFP would be expressed wherever the
original gene would have been expressed Would this work?
Also again, how do you know where it is without testing
every tissue in the body? Once you know where the gene is,
would you then do a Western blot, or is the Western blot
INSTEAD of the protein tagging?
I was also wondering what is the best way of testing the
function of a gene product? Would it be to KO or knock down
the gene and then maybe test that you knocked out the
correct thing by doing a rescue experiment? Is that the
best way of testing that a KO experiment had worked or
would qtPCR be better?  Also for a gain of function
experiment, what would be the best way of testing that
worked? What qtPCR be good or could you use a comparative
hybridization microarray between the tested tissue and a
control?
Thank you once again,
Gemma

Answer
Hi Gemma,

Sorry for replying late to this!!
Pl. find answers to some of your questions below:

1. What is the best way of testing where a protein is
expressed? how would you find out where it was expressed without having to extract tissue from every cell in the body??

The best way would be to use an electron microscope where the protein of interest-X is detected with an anti-X antibody labelled with dense material such as gold. With electron microscope you can see individual organelles and then know in which organelles protein X is localized.

2. So the GFP would be expressed wherever the
original gene would have been expressed Would this work?
Also again, how do you know where it is without testing
every tissue in the body?

The second best way would be to use fluorescent or confocal microscope to see where the protein is when it is tagged to GFP. Since you cannot actually see the organelles in confocal microscope, one actually uses other standard makers. For example, to label a Golgi you use antibody againt the standard golgi protein GM130. Now you use anti-GM130 that is already tagged with flourescent dye such as FITC. So your Golgi will glow green under micrscope. In the same section you use anti-X antibody tagged to another dye such as cy3 which is red. Therefore if X is localized in Golgi you will see green and red together.


3. or is the Western blot
INSTEAD of the protein tagging?

Western blot is done normally in cases where the protein is not tagged.


4. Although Knock-out will be best, however Knock-out will be equally well and a standard qPCR can be used to know the expression.

Hope this helps,

Best,