Hi I am trying to developing indirect ELISA using antibody and recombinant protein (still finding).
For protein stadndard I found some recombinant protein product from variety manufacture, but I saw that some of their product is not full length product. Some product has only half of their original size (25kD original size but the product is only 11kD).
In this case, is it same amount when same mass of these protein fragment or partial protein (I am sorry, I don't know what to say) and full length protein was used.
I meand when I load 2ug of these protein fragment and full length protein, could I get same signal in ELISA? or Could I use these partial protein as my protein standard for ELISA?
As long as your control has both epitopes on it, you should be able to use it as a reasonable standard. (However, there are always drawbacks of using any standard that is not 100% identical to your target -- but this kind of problem would apply to any standard you would choose). When you are starting experiments like this, it's more important to make sure you get a reproducible (and preferably linear) response to the concentration of your standard (peptide or whatever else you use).
The calculation should not be in ug (mass) - but in molar concentration (moles/volume). If you convert to moles of standard, you will be able to correlate the moles (or molar concentration) bound of your actual experimental samples, and you can calculate back to ug/mL if you prefer to look at your data that way.
As long as your control has both epitopes on it, you should be able to use it as a reasonable standard. (However, there are always drawbacks of using any standard that is not 100% identical to your target -- but this kind of problem would apply to any standard you would choose). When you are starting experiments like this, it's more important to make sure you get a reproducible (and preferably linear) response to the concentration of your standard (peptide or whatever else you use).
The calculation should not be in ug (mass) - but in molar concentration (moles/volume). If you convert to moles of standard, you will be able to correlate the moles (or molar concentration) bound of your actual experimental samples, and you can calculate back to ug/mL if you prefer to look at your data that way.
Hi there,
2µg of a 25kDa protein contains around 2-fold less molecules than 2µg of the 11kDa. If both proteins contain the epitope recognized by the anibody used in ELISA, the 11kDa protein should give twice the signal obtained with the 25kDa if working with the same mass of proteins.
To Gelfand, I never thought about molar concentration.
For example, there is 10kD of partial protein and their original size is 22kD.
The 10ng/ml of partial protein was used in standard (1nM, from convertor), and my sample has same signal with it. In this case, my sample has 1nM of protein and this means that it has 22ng/ml of concentration not 10ng/ml.
Is it right?
10ng/ml or partial protein = 1nM
--> The sample has same signal with it = sample has 1nM of protein concentration
----> The sample has 22ng/ml, NOT 10ng/ml.
(The mass concentration is converted from molar concentration
based on their original size, not fragment size,)
To Liger, thank you for your continuous answer.
That is what I am worried about.
I found a full length recombinant protein (It looks like, but I am not sure, I have to find the full length "aa sequence" of protein and check it.), but it is too large mass and expensive. The small mass one from other company has not full length protein.
I am totally in agreement with the explanations given by Craig and Dominique
Sorry to late.
To all of you thank you for your kind answer.
It is really helpful for me.
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