I am trying to study the oligomeric state of peptide of size ~3kDa with theoretical pI of 10.19. I have tried using 15% SDS-Glycine system and done silver staining, but no bands visible. Then, I tried with 16.5% Tris-Tricine SDS gel followed by silver staining as well as coomassie staining, I got smear near the end of gel and not crisp bands. I repeated the same with 16.5 % Tris-Tricine Native PAGE, by reversing the anode (since its a basic peptide), but the sample didn't go into the well. So I ran normally, it ran, but I saw some precipitate near the edges of well. I think that my peptide didn't enter the well itself.
Could anyone help me how to study the oligomeric state of peptide and suggest an appropriate gel system / method for the same?
http://books.google.com.br/books?id=5n5oho-S1psC&printsec=frontcover&hl=pt-BR
Chapter 8 ;)
Do you expect your peptide to oligomerize via covalent interactions? Because SDS should theoretically disrupt such linkages. Also did do you compare reducing or non-reducing SDS-PAGE profiles? although I am not sure if it would be of any help!
>Generally Glycine SDS-PAGE system may be suitable if your protein conformation state of interest is in the range of 250 to 10 kDa. For small weights (Also my experience with this methodology shows that diffuse peptide bands in the low molecular weight can result in pre-cast, ready-made gels if they are too old. So it may help to do it on a fresh gel, or in gels prepared in-house.
>Is the peptide oligomeric state stable at low pH? use of MS too may help, since it will give you exact weight of the peptide. But this may require sample analysis under acidic conditions, and this method would not work if the oligomerization state changes.
>As Oscar said, analytical ultracentrifugation may be useful!
Thanks for your kind reply.
I have used fresh gel made in-house only. But, I got smears only for the sample in the gel and sharp band for the marker. Could you suggest some ideas regarding the same?
Also, could anyone tell me the procedure for analytical centrifugation method?
Thanks for the additional information! It is hard to determine the exact cause for precipitation.
The sharp marker bands indicate that your running buffer and the gel reagents were fine and you may consider the sample buffer or the sample for further investigations. Precipitation or less distinct bands can also appear due to high ionic strength of the sample. Acid precipitation of the sample and washing followed by re-suspension in buffer to reduce ionic strength has been suggested.
Your results indicate that protein may have been in an oligomeric state in Native page but may have got disrupted in the SDS-PAGE (Tricine gel). I am sure you appreciate that the SDS would disrupt all non-covalent interactions.
Hope this helps! Unfortunately, I don't have experience with analytical ultra centrifugation.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics