Hi everyone
Its a small protein with 5.7kD weight. After doing weight adjustments considering acidic amino acids and also 3XFLAG tag, it should be 11.6 kD.
But on 16% tricine gel, it appears between 15kD and 25kD (almost 20kD).
What could be the reason?
I have checked it in cell extract and also after FLAG purification.
Thanks.
Could it be glycosylated? If there is N-linked glycosylation, treating with PNGase F to remove it should result in the protein migrating at the expected mol. wt. for the polypeptide.
Is it possible for your target to be dimerized? Do you able to confirm the observed mass at any MS system? Small proteins can also be present as conjugated in the matrix, therefore co-purifying a prey protein may have occurred. A highly glycosylated target is another option, you may confirm this by glycoproteome staining methods.
Adam B Shapiro Thanks for your reply. At the moment, i do not know if it is the case. but i can definitely try this PNGase F to check it.
İsmail Emir Akyildiz Thank you for your answer.
As its a hypothetical protein and nothing is known about it yet.
It could or could not be a dimer. Last protein that i was working on was also a dimer but i could see 2 bands on the gel instead of band shifting.
Can you please suggest which staining method is best to confirm glycosylation?
Pro-Q Emerald 488 glycoprotein stain or traditional PAS method would do the job. You may select according to your detection preference...There are kits to be purchased or in-house developed reagents that can be used for an easy application. If you have an HR-MS system, you do not need any application such as PNGase treatment or glycoprotein staining. Deconvulated spectra potentially describe the glycosylation and exact mass, charge profile, PTMs and other unknown attributes for a hypothetical protein.
Good luck
Try to purify it. As it will make sure that some other molecule must not be interacted with this protein.
Others have already given you the most likely reasons for the discrepancy, and I have a few of my own. Try optimizing the following if you have time.
- The resolution of tricine gels can vary, and sometimes the separation of proteins in the 15-25 kD range may not be as sharp as in other regions of the gel. The migration of proteins can be influenced by the gel percentage and the running conditions.
- The apparent molecular weight of a protein on a gel can also be affected by its conformation. Proteins with highly folded or compact structures may migrate differently than their expected molecular weight. This is especially true for small proteins.
- Small proteins can sometimes aggregate or form complexes, which can affect their migration on the gel. Ensure that your protein is properly denatured before loading onto the gel. Calibrate your gel using known molecular weight markers to verify the accuracy of the molecular weight estimation. This will help you confirm if the gel migration pattern is consistent with the markers.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA