You could try the covalent fluorescent dyes (Cy...) which are used in 2D-DIGE. They are amino reactive and obviously will increase the MW, but not affect the charge.

Hello,

I wonder if it would be easier to show that the dipeptide is retained with anion exchange chromatography, but not with cation exchange chromatography. That way you could detect the presence or absence of the dipeptide in the elution fraction directly with spectrophotography. If you have access to an HPLC and an ion exchange column, you might even be able to do this in one step by comparing the retention times.

I am not fully convinced that you can see the dipeptide in gel electrophoresis due to its very small MW. It is a well-known issue you need to fix the peptide before staining, you may investigate this idea through tris tricine gel electrophoresis for small proteins and peptides...Look for the glutaraldehyde fixation process prior to CBB stain...On the other hand, the most reliable way to validate that your peptide is negatively charged is mass spec infusion mode analysis..-ESI definitely clarifies if your peptide is negatively charged...

Besides IEX is also a good option but dipeptide probably lacks chromophores, therefore you better combine IEX with a conductivity detector rather than conventional UV detection...(look for ion chromatography applications for this idea)...

As Wolfgang Schechinger mentioned, there are dyes could bind the N terminus of your dipeptide via the isothiocyanate group at neutral pH ranges, e.g. FITC. FITC, however, is negatively charged at neutral pH.

You could also order peptides with a fluorophore from a commercial company, but they are costly.

Also, silver staining is a known technique for SDS-PAGE, but the SDS detergent itself inherently gives a negative charge to your peptide.

Thank you Sir for your suggestion. I could have tried with positively charged cyanine dyes but currently, we dont have the facility of 2D-DIGE. Wolfgang Schechinger

Ryan Wheeler Thank you for your suggestion. I can try IEX chromatography but since my peptide doesn't have a chromophore so impossible to detect using a UV detector in our conventional HPLC. Have to look for another option. But the suggestion is indeed thoughtful.

Sabnam Kar You don't need 2D-DIGE equipment. This was just an example for the use of these Cy-dyes and intended to help you to identify them. After labeling your peptide, you should be able to visualize it in your experiment as you have described it, just by using a transilluminator with a suitable spectrum (e.g., blue or UV for dyes with a fluorescein like spectrum).

To visualize a negatively charged dipeptide in agarose gel electrophoresis, you can use a negatively charged stain such as Ethidium bromide, which will interact with the negatively charged dipeptide and show up as a distinct band. Alternatively, you can try using a silver staining protocol such as the one described in the method of Gomori, which is sensitive enough to detect small amounts of protein or peptides.

However, agarose gel electrophoresis may not be the best method for visualizing a small charged peptide, as the resolving power of agarose gels is generally limited compared to polyacrylamide gels. If possible, you may want to try using a polyacrylamide gel with SDS-PAGE, which can provide better resolution for smaller peptides.

In terms of concerns with your current method, it is possible that the dipeptide has migrated out of the gel during electrophoresis, or that it was not loaded into the gel properly. Additionally, if the dipeptide is highly charged, it may have a tendency to interact with the gel matrix or other components of the running buffer, which can affect its migration. To minimize these issues, you may want to ensure that the dipeptide is properly dissolved and well mixed with the tracking dye prior to loading it into the gel. Additionally, you can try modifying the running buffer to optimize the migration of the dipeptide.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

Thank you Nikolay Klyashtornyy for the detailed response. We actually tried both agarose and SDS page but we could not spot our peptide in gel. So could not get the any useful data out of it.

You may try radiolabeling which involves incorporating a radioactive isotope into the dipeptide molecule, which can be detected using radioactivity measurement techniques.

There are different approaches to radiolabeling dipeptides, depending on the specific goals of the study. One common method is to incorporate a radiolabeled amino acid during the synthesis of the dipeptide. For example, you could use a radioactive form of an amino acid, such as ^3H-leucine or ^14C-alanine, which would be incorporated into the dipeptide during its synthesis. The radioactivity can then be detected using techniques such as liquid scintillation counting or autoradiography.

Radiolabeling allows for sensitive detection and quantification of dipeptides. It is particularly useful in studies involving the metabolism, transport, or degradation of dipeptides, as well as investigations into their cellular uptake or receptor binding. By using radiolabeled dipeptides, researchers can track the fate and distribution of the dipeptide molecules in biological systems.

It's important to note that the use of radioactive materials requires proper handling, safety precautions, and compliance with regulatory guidelines. Additionally, alternative detection methods, such as fluorescent or mass spectrometry-based techniques, are often preferred due to their ease of use, non-radioactive nature, and higher throughput capabilities.