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relationship between protein size, lysate concentration, and band intensity

western blot

Best Answer bob1, 25 November 2013 - 07:29 PM

There isn't any general rule for proteins, as normally these sorts of things don't happen.  I'm guessing that you have a series of truncation mutants for the same protein and are probing them all with the same polyclonal antibody raised against the full length protein.  If this is so, then there might be a relationship between the intensity of the bands and length, but you would have to establish that for yourself, which you could do you plotting band intensity versus size, or amount of protein loaded, or some other factor, and see if there is a relationship.

 

As these are transfected samples, I don't see why they are precious, surely you have more plasmid DNA (or can make some more -glycerol stocks are your friend), and 293's are a dime a dozen. 

 

The DNA thing is due to the number of molecules of EtBr that get incorporated into a strand - this happens at a defined amount per helical turn, so the shorter a fragment, the fewer molecules get incorporated and the less intense it seems.

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#1 nocic

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Posted 25 November 2013 - 06:04 PM

I've successfully transfected series of vectors encoding wide range of protein sizes into HEK293T cells ( they are not cotransfection, instead several different transfections). After harvesting and determining the lysate concentrations, I load the same amount of proteins into SDS-PAGE gel, probe (they all use the same primary and secondary ab), and visualize. My results showed that the biggest protein (~120kda) had the most intense band, while the rest of my samples were decreasing in band intensity as I go from highest to lowest protein sizes (all the way down to ~6kDa).This is something that I've observed while working with DNA.

The size of my observed proteins of interest are as follows:
~120kDa (very strong band)
~40kDa
~30kDa
~29kDa
~10kDa (very faint)
~6kDa   (barely visible)

They all need to be on the same gel and the goal of my next repeat experiment is to show all bands in relatively equal intensities. I plan on doing this by lowering the amount of lysate I put in for visualizing the 120kDa protein while increasing the amount of lysate for the rest, by 2x, 4x, 6x, etc. However, these are precious samples so I cannot perform many series using trial and error alone.

Is there a general rule that I can follow? (For example, 10ug of lysate/10kda protein of interest and 1ug of lysate/100kDa protein of interest yields relatively comparable band intensity)


Edited by nocic, 25 November 2013 - 06:32 PM.


#2 bob1

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Posted 25 November 2013 - 07:29 PM   Best Answer

There isn't any general rule for proteins, as normally these sorts of things don't happen.  I'm guessing that you have a series of truncation mutants for the same protein and are probing them all with the same polyclonal antibody raised against the full length protein.  If this is so, then there might be a relationship between the intensity of the bands and length, but you would have to establish that for yourself, which you could do you plotting band intensity versus size, or amount of protein loaded, or some other factor, and see if there is a relationship.

 

As these are transfected samples, I don't see why they are precious, surely you have more plasmid DNA (or can make some more -glycerol stocks are your friend), and 293's are a dime a dozen. 

 

The DNA thing is due to the number of molecules of EtBr that get incorporated into a strand - this happens at a defined amount per helical turn, so the shorter a fragment, the fewer molecules get incorporated and the less intense it seems.



#3 nocic

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Posted 25 November 2013 - 07:40 PM

They are mutant fragments indeed but I'm probing them all using anti-flag (the vector contains 3x flag). The reason why I said they are precious is because I would like to finish before the holidays so I don't have time to do another transfection ;D

 

Thanks for the clarification regarding DNA.

 

Thanks you so much! You gave me more confidence for my repeat experiment!


Edited by nocic, 25 November 2013 - 07:42 PM.


#4 bob1

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Posted 25 November 2013 - 11:22 PM

In that case, your intensities are solely due to expression levels, and could well vary with repeated transfections. 







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