Few Questions About Cell Biology

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Offline zetx

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Few Questions About Cell Biology
« on: 15/04/2009 08:52:08 »
I have 3 questions regarding about cell biology area.

Q1: A number of human cancers are accompanied by single amino acid substitutions in a protein called Ras. If you are given tumour protein samples from 5 different patients diagnosed with cancer and a control from  a non-cancerous individual, which of the following techniques is best suited to start off your analysis to determine if the Ras protein has been mutated in any of the samples.

The answer for this question is "Two dimensional gel elctrophoresis" but i am not sure why that is the case.

Q2: How many of the following proteins would pass through the RER?
actin, p21CIP, p53, insulin, cyclin, p34(Cdc2); histone HI; LDL recpetor; a clathrin antibody

The answer is 3 but i can't seem to figure out which.

Q3Tubulin like actin has many isoforms and the amount of each varies between cell-type. There are at least three tubulin isoforms and you would like to purify each in order to make a monoclonal antibody (mAb) that specifically recognizes only the gamma isoform. WHat approach below would be the best place to start to get enough gamma isoform to inject into a mouse for mAb production?

I thought it would be DNA microarray analysis but in the exam, it says none of the above - so what kind of method would be used to determine this?

« Last Edit: 15/04/2009 09:08:30 by zetx »


Offline WylieE

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Few Questions About Cell Biology
« Reply #1 on: 24/06/2009 05:03:47 »
 I'll give question 1 a shot.

 I'll give a brief description of Two dimensional gel electrophoresis (I'll abbreviate it 2D-GE) first then explain how it fits to answer the question.  2D-GE is used to separate out proteins by their physical structure.  With 2D-GE proteins can be separated by two properties, the idea is that most proteins should have an individual "signature" when you compare them by two criteria. 

A simple comparison would be if you are having a party with 100 people and you'd like to take a picture from above with everyone in their own spot in a reproducible manner. If everyone was lined up along a wall by height. You might have 5 people that are 5'11" so if you asked them to line up again later the 5 people would be in the same general spot, but they might not be in the exact same order.  However, if you also separated each of these height groups by how much each person weighed, by having the people move away from the wall a certain distance based on their weight, you could now take a picture from above with most people would be in their own spot in the room and you could get them back to the same spot again later.

  The same applies to proteins.  All the proteins from a sample are collected, and then these are separated in one dimension.  Commonly, this is by isoelectric point - but other measures could be used to separate the proteins (just like you could use other measures to separate the people, hair color, salary etc.).  Then the gel is turned 90 degrees and the proteins are separated by size.  This should give most proteins their own unique spot on the gel.

Now suppose at your party someone has taken advantage of your hospitality and has eaten all of your lasagna before you put it out for everyone else (pretend lasagna is really, really heavy).  If you lined everyone up again and took a second picture you could compare this to the first and see who moved relative to their original position.

So for RAS, an amino acid change can change the isoelectric point, how RAS interacts with other proteins, or other physical properties.  If you compare a control sample to the 5 patients you could look and see if the position of RAS moved relative to its position in the control sample.

All that said however, I think it might be cheaper and easier to amplify RAS DNA from each of the samples and sequence it to answer this question.  I guess the advantage of the 2D gel is that if RAS wasn't what changed, you might be able to identify other proteins that did change.  But if you really wanted to just know if RAS changed, I don't see how a 2D-GE would be better than sequencing the DNA.