how do we know ......???

[Joumana]

Under the microscope there's a protein in the membrane...

 

How will I know weather this protein has been on the membrane since it delivery

or has been recycle to the cell and back to the membrane ???

 

 

 

I hope to found an answer ^^

 

All My Regards

 

JOJO

[CharonY]

First, how did you detect the protein in the first place? Fluorescence tagging? Antibodies? Second, what precisely is your question? Whether it is a membrane associated or integral membrane protein versus co-purified membrane fraction?

[Joumana]

this question l was asked by a doctor ,and the question wasn't based on observing protein under microscope ... ( and we are pre-student ) ... so he just look for the idea

 

about which microscope it's Fluorescence tagging.. (because he was discussing it

[CharonY]

Sorry, maybe it is just the lack of caffeine in my blood system, but you are not making terrible sense to me right now. First you started off with

Under the microscope there's a protein in the membrane...

(my bold) and now it is not based on microscopic observation but based on... what?

 

Also the main question is still not clear to me. Evidence for e.g. localization can be based on different techniques, of which some are considered more definite than others. Thus the actual technique in question is significant.

[Joumana]

doctor said:

Here is the question:

Let's say you are visualizing a fixed cell and you stained for a certain protein. You found this protein on the cell surface (i.e. on the plasma membrane).

This protein may have just been delivered to the plasma membrane (coming from the golgi), or it may have been at the membrane, recycled back to the inside of the cell, then recycled back to the surface.

So, if you find a protein on the cell surface, how will you know whether it has been on the surface since it has been delivered there, or it has gone to the membrane then to the inside then back to the membrane?

Edited September 15, 2009 by Joumana
Consecutive posts merged.

[CharonY]

OK, that makes more sense. Note that I am not an expert in this field, but assuming that he/she meant an antibody staining after fixation, there is no real way to confirm origin as (afaik) the recycled proteins do not undergo any specific and detectable modifications. Additional experiments are needed and may either include disruption of recycling pathways or tracking the internalization of (labeled) surface proteins. For instance, if the protein in question is not stably localized in the membrane, one could label it shortly (e.g. with an antibody), wait until it vanishes and check for the re-appearance of the antibody on the surface after re-incubation.

[jimmydasaint]

I agree with CharonY here. It sounds like you would need to perform cell fractionation following the radiolabelling of in vitro cultured cells with 15-N, for example. By careful, stepwise cell fractionation, you could track the radioactive cell fraction from ribosomes ---> Golgi --->Golgi vesicles ---> cell membrane, and further if necessary. Alternatively using the anti - protein antibodies (monoclonal preferably), you could perform immunoprecipitations of cell proteins that have internalised 15-N or similar isotopes and do a 'time line' again. Remember that background 'noise' will be filtered out by using monoclonal antibodies with a tight specificity for the membrane protein.

[Joumana]

Thank you Mr.CharonY

& Mr.Jimmydasaint

 

Thank you alot ..

[GDG]

doctor said:

 

Your solution is to tether the protein to a substrate. Hard to internalize if it is chained to the desktop

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Merged post follows:

Consecutive posts merged

At least, if you are trying to insure the protein stays put, you can do that.

 

If you are trying to determine whether or not the protein naturally cycles between the cytoplasm and membrane surface, you can tag it with GFP (green fluorescent protein) and watch it under a microscope. See, e.g., C.C. Lanning et al., J Biol Chem (2003) 278:12495-506 (free online article).

 

IIRC, there are also GFP constructs that are sensitive to cytosolic pH or Ca++ concentrations, so that you can tell if the protein is in the cytosol simply because that is the only place it will light up.

 

Or were you asking something different?

[CharonY]

Problem is that in the proposed example the cells is already fixed. At this point dynamic observation are out of the question already. Even if this was not a problem, the question appears to be aimed at identifying recycled proteins.

Individual GFP constructs are hard to follow for a longer time (i.e. a full cycle). And presence of a protein in both cytosol as well as membrane is still not an indicator of recycling. Of course, this is how I understood the question, which may be wrong.