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the intensity ratio between light chain and heavy chain on SDS-PAGE


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#1 wenchien

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Posted 01 November 2012 - 05:59 PM

Does anyone know the idea intensity ratio between light chain and heavy chain of one antibody on SDS-PAGE?
And what kind of reason that change the ratio?

#2 John Forsberg

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Posted 01 November 2012 - 06:19 PM

What detection method are you using for visualizing the antibody chains?

Using secondary antibodies on Western blots will vary wildly on how well they bind to the heavy and light chains, depending on whether the secondary antibody is a monoclonal or polyclonal and where its epitopes are. I wouldn't expect quantitative measures of heavy:light using antibodies.

For Coomassie staining, assuming an equal amount of staining per gram of protein for the heavy and light chains, I'd guess offhand that the ratio should be about 2:1 heavy:light, as the heavy chain is usually around 50 kDa, and the light chain is usually around 25 kDa (forgive me if my numbers are a bit off, I don't remember exactly). This is also assuming that the staining is linear in the concentration range of the proteins in the gel (I'm not sure offhand if this is the case, although I suspect it probably is). Coomassie binding may be somewhat sequence-specific also, but I'm not sure how much it varies.

#3 wenchien

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Posted 11 November 2012 - 07:51 PM

Hi john,
thank you for your reply,
I try bothe silver stain and SYPRO RUBY staining to develop the page.
My idea is to see 2:1 ratio on the PAGE
but Unlike the intensity of heavy chain, the intensity band of light chain is very weak in SYPRO RUBY and barely can not see when developing with silver.
I was wondering what cause these phonemonem
Another problem is the band of light chain shows smere instead of "one band" shape of heavy chain
At the begining, we thought it might cause by degredation and we took the new rabbit IgG but get same result.
Is there any other possibilty to cause these?


You mentioned about the mAb and pAb and where its epitopes are will affetc the binding of heavy and light chains.
what kind of result will I get when I use mAb and what in pAb?

Kindly appteciate your help
wen

#4 John Forsberg

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Posted 12 November 2012 - 07:52 AM

I'd forgotten that antibodies are glycated, so you may see variation in their position on a gel based on the extent of glycation. It could be that the light chain is more often modified and that's why it's more of a smear?

Perhaps the glycation also interferes with the Sypro Ruby binding? I'm not sure. Hopefully someone else can reply with more information.

For comparing monoclonal antibodies versus polyclonal, I would expect that polyclonal secondary antibodies would show stronger bands for both chains, since they should bind to multiple sites on both the heavy and light chains, whereas a monoclonal will only bind one site (either heavy chain alone or light chain alone).

I'm not sure there's a way to ensure you see a 2:1 intensity ratio on a gel.




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