Dear Experts,
Is there recommended protocol for hydrazide-dye labeling the antibody/protein?
To my understanding, labels with "hydrazide" could use to label the aldehyde "-CHO" group of antibodies in Fc region.
However, couldn't found a suitable protocol to perform the test.
Most of the resources found on the website highlights the mechanism but lack the required material (chemicals to initiate reaction, such as 2-IT is thiolated antibody) list and step
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/carbonyl-reactive-crosslinker-chemistry.html#2
Could you please help to share the information?
Dear Po-Han Chao,
to obtain aldehydes from carbohydrate side chains, you may oxidize them with sodium metaperiodate. Then quench excess reagent with glycerol and remove byproducts by gel filtration (e.g. with PD10 columns), or try it quick and dirty by adding the hydrazine directly after the oxidation, and then quench excess periodate before you go onto the column (otherwise, the excess periodate will oxidize the Sephadex and cause losses by making the antibody bind to it as imines via the lysines). The latter version might have higher yields, as you'll only have one clean-up.
For an easily adaptable protocol, check the antibody/peroxidase labeling chapter in "Harlowe and Lane - Using Antibodies". You should find the book in your library. Also "The Hermanson" which is referenced in your link should have a suitable protocol.
Dear Po-Han Chao thank you for posting this interesting technical question on RG. In contrast to Wolfgang Schechinger who gave a very good expert advice we are no specialists in this field. In think that the basic chemistry behind this is already outlined in the link which you provided together with your question. In addition, it might be of interest to you to have a look at the following useful link:
Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide
Article Fluorescence labeling of carbonylated lipids and proteins in...
This paper has been posted by the authors as public full text on RG. Thus you can freely download the paper as pdf file. In particular, please check the references [3] and [13]-[26] cited in this paper. They refer to the characterization methods of carbonylated proteins.
Good luck with your research!
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