My protein having his tag which I have purified using Ni-NTA chromatography. I want to use purified protein for Cell culture studies on Macrophages.
To study entry of protein into the cell, I want to label my protein with fluorochrome to visualize under fluorescence microscope.
Kindly help.
Hi Parul Bhatt . You need to choose which dye best fits your needs and instrumentation. There are tutorials for that:
https://www.thermofisher.com/is/en/home/life-science/cell-analysis/fluorophores.html
https://www.thermofisher.com/is/en/home/life-science/protein-biology/protein-labeling-crosslinking/fluorescent-protein-labeling.html
Dear Parul
Once you choose the fluorophore you can find several commercial kit to label protein of antibodies with fluorophores.
eg;:
https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0019835_AlexaFluor_ProteinLabelingKits_UG.pdf&title=VXNlciBHdWlkZTogUHJvdGVpbiBMYWJlbGluZyBLaXRzIChGb3IgQWxleGEgRmx1b3IsIFBhY2lmaWMgQmx1ZSwgRmx1b3Jlc2NlaW4tRVgsIGFuZCBPcmVnb24gR3JlZW4gNDg4KQ==
https://biotium.com/technology/antibodyprotein-labeling-kits/
https://www.abcam.com/nav/primary-antibodies/antibody-and-protein-labeling-kits
different kits may have different reactive moieties and therefore reacts efficiently with different residues (eg primary amines, cysteines, glycosylation) of proteins to form stable dye–protein conjugate,
In some unlucky case if the labelling happen in a functional residues is possible that the labelling will affect the protein functionality and therefore if you have in your hand a functional assay is every better to check if the labelled protein is able to maintain its functionality in vitro before use it in complex experiments to avoid false negative results.
ciao
Manuele
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