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Designing experiment - (Dec/08/2012 )


I am expected to design an experiment like the one below. Can you help me find my way to design this experiment? (in means of molecular biology techniques)
I am not so familiar with the techniques since I graduated from chemical engineering. Thanks!

You are working on Interleukin-6 signal transduction pathway for inflammation in cell culture system. When you add Interleukin-6 to cells expressing Interleukin-6 receptor, 100 kDa protein is highly phosphorylated on tyrosine residues. Please use all required advanced molecular biology techniques in order to answer the questions.

a) How do you clone gene encoding this protein, show genomic and also cDNA of your gene encoding 100 kDa protein?
B) You showed that this is new transcription factor and please show this transcription factor activates some of genes.
c) Please show location of this protein in cells before and after activation.
d) How do you show this protein binds to promoter region of gene (Please put control and make sure this protein specifically binds to this region)?
e) How do you show that which base is important for binding to your transcription factor?
f) How do you show other protein binds to your transcription factor activates gene expression?


As this is obviously homework I have moved this to the homework section / molecular biology section of the forums.

How about you show that you are making some effort towards answering these for yourself, rather than us just providing answers. It will help you learn better.


For the question (a) I am thinking about PCR. reverse transcription- PCR for cDNA cloning.
For (d), I am thinking about EMSA.
For (e), maybe a kind of mutagenesis can be applied.
And maybe for ( b ), Western Blotting.

Anyone agrees with me?


Are these supposed to be a questions on what general methods would be used for each task?
(I have sometimes problem with these virtual experimental designs as homework questions, because sometimes they really lack the meaning behind a question, for example the question about cloning in real setting would need to include what would be the purpose of a clone, wha would anyone wanted to clone DNA and so on)

But in general, a) yes, you need PCR/reverse transcription-PCR but for the complete cloning also.. well the cloning itself which requires other basic methods
The first question is not clear for me, does it have two parts? Like the first - "what to use for cloning" and the second - "show genomic and cDNA" (sequence I presume), I don't really get what "show" means, does it mean how can you find yout the compltete sequence? There is a specific method that can tell you the sequence (after PCR). From the practical point of view, the lenght of the protein and logically the corresponding DNA/cDNA has to be taken in account too (you can look up average weight of an amino acid to get idea how long your protein is and coding sequence will be). But as I say, that's the problem with these questions as I don't know how much detail is required (maximum detail is required in real life).

For d) EMSA can be used I guess, but there is one other general method coming to my mind first, that can tell you whether certain region(s) interacts with a specific protein, can you think of it? It's advantage is you can test more proteins and more regions at once.

e) I agree

As for b> Western blott will show you presence of a protein in a lysate and it's amount, specific antibodies can be used to detect phosphorylated form only (and I wonder what the question about this was left out as this is probably the first thing you would check, if it's phosphorylated), so your gene is a transcription factor, so you actually want to measure transcription of his targets, so....
You could detect the targets as proteins by WB, but do you think there is always an exact correlation between rate of transcription and amount of protein?

c) I have an idea fro this one, still having in mind the antibodies specific for a protein form.. and of course you need something to watch the cells in..