Dear Seyedeh,

Attached please find two publications that cover the answer to your question.

PROTOCOL

Tricine–SDS-PAGE

Hermann Schägger

Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, Haus 26, D-60590 Frankfurt, Germany.

Correspondence should be addressed to H.S. ([email protected]).

Published online 12 May; corrected online 10 August 2006; doi:10.1038/nprot.2006.4

Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine–SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second

dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine–SDS-PAGE, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1–2 d.

Hoping this will be helpful,

Rafik

Hi rafik

thanks for reply

Dear Zahra

Have you considered working with higher concentrations of your sample or going for purification steps and accumulating runs in order to increasing the abundance of your polypetide?

Also do you know if your polypeptide has hydrophobic properties in this case you should consider specific heat traitement (30 min at 50°C) before loading into your gel so your hydrophobic polypetide will migrate properly.

You should also pay attention into your staining and destaining method. I particularly appreciated using the PhastGel Blue R dye (GE,Healthcare) wish i destain after an overnight staining, using a 30% methanol, 10% acetic acid in water solution.

Another thing which i found useful is to use the laemmli loading buffer rather that the Tricine protocol loading buffer some how it gave me better results.

Also try to use the Spacer gel method included in the Tricine SDS Page paper. this gel helps to concentrate and sharpen your protein bands so even if they are too tinny they will be more visible.

I hope this can help you. Please feel free to contact me if y have any further questions.

Best Regards

Ibtissam

Dear Ibtissam

Thanks for comprehensive answer. I examined higher concentration of samples and treated them (5 min at 100°C). But the biggest problem is bad resolution of gel. I mostly focused on tricine SDS-PAGE and always used tricine laoding buffer and I don′t have enough knowledge about  the Spacer gel method. finally I repeat my question to you: do you have any protocol that can show these peptide on SDS-PAGE gel?

Thank you

Dear Zahra

can you send me images of your gel so i can see exactly the problems you are having with your sample.

I would appreciate if you can send me an email address where i can send you the protocols i used and also explanation about the spacer gel method.

attached to this response is a paper where i used Tricine SDS Page for the resolution of small polypetides of arround 6-7 kDa. You can see on last gel figures the result of using a spacer gel and also you can refer to the protocol i used.

And again let me attract your attention to the heat treatment as the 5 min at 100°c treatment dosn't work for hydrophobic protein. 

Kind regards

Ibtissam

Dear Zahra,

We successfully characterized a small fragment of GroEL using Tris-tricine SDS-PAGE to recover the fragment:

http://www.ncbi.nlm.nih.gov/pubmed/8559246

In the work quoted above, the method by Schagger described above, in Rafik's response, was used to carry out tris-tricine SDS-PAGE.

Here is the link to the original paper by Schagger et al:

http://www.ncbi.nlm.nih.gov/pubmed/2449095

I would use the Nature protocol under Rafik's response since it is more updated. 

Just wanted to let you know that Tricine does work very well for separation of small (

Dear Hediye

thanks for reply.

I studied Nature protocol several times but I need a set up protocol. I tried to use several protocol and didn't see 5 and 3.4 KDa bands. I know about Tricine and as I described above the weight of peptides about 2.7-7 KDa.