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isoforms in cell line

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isoforms in cell line

Postby vlad81 » Sun Jun 06, 2010 7:05 pm

Hi all,
I am working with cell line and looking at a particular protein which has 25 protein coding isoforms from a wide range of molecular weight.

Can anyone tell me if there is some max possible isoforms co-expressed in one particular cell line or is it really possible to have all of them, 25, in only one cell lyne?

My western blots look like zebra stripes and I am quite confused what to explain to my supervisor.

Any help will be appreciated.
:?
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Re: isoforms in cell line

Postby JackBean » Sun Jun 06, 2010 8:08 pm

vlad81 wrote:I am working with cell line


LOL :lol: really? :lol: :lol: :lol:

Are these 25 isoforms predicted or even better confirmed? Or is that just your guess because of the blot?

My guess would be unspecific binding, maybe some degradation products.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: isoforms in cell line

Postby biohazard » Mon Jun 07, 2010 6:29 am

JackBean wrote:
vlad81 wrote:I am working with cell line


LOL :lol: really? :lol: :lol: :lol:



I do not quite understand what is so funny in this? After all, he could have been working with tissue lysates, serum/plasma or tissue culture supernatants and study the proteins in there...

Anyway, sounds bit odd that your antibodies would bind such a big range of molecules. Although if your target protein has some variable region or odd glycosylation patterns and your antibody only targets the "constant" region, extra bands might appear. But if the cell line is truly homologous, I doubt what you see is really all the protein isoforms.

Is the protein intracellular, membrane-bound or secreted? Some extra information on this protein might help, with this we cannot say much.
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Postby vlad81 » Mon Jun 07, 2010 1:18 pm

thanks for the response guys,

I understand that may question may well be funny, but I am affraid my supervisor is not going to laugh as much when I show him my blots.

JackBean, I found the isoforms for the protein on http://www.ensembl.org/index.html. For 16 of them it is written " Protein coding", for the rest - "No protein product". However, on NCBI you can find only 8 of them.

I used two antibodies from two different companies, but the result is the same.

Biohazard, the cell lysate I have used is from hypotetraploid cells with average chromosome number 84. the protein is a transcription factor with MW, with respect to the different isoforms, ranging from 2-55kDa. However, most of the isoforms are about 50-55kDa and yes they have glycosylation sites, so they would appear at ~60kDa.

thanks a lot!
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Postby JackBean » Mon Jun 07, 2010 7:40 pm

the question by itself was not funny. The sentence I've cited was, as one would expect info about the cell line... but biohazard is right... (damn! :lol:)

the question is not, whether it is protein coding or not, but whether there are any evidence for such splicing variants.
http://www.biolib.cz/en/main/

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