If you want to look for a dimer I guess you shouldn't denature the protein. Maybe I don't really understand what you are trying to say.

You might want to use biophysical methods, such as static light scattering, to determine the molecular weight of your molecule in the presence or absence of lipids. If it dimerizes you should get a molecular weight of twice the size of the monomer in the presence of lipids....

In fact, this sequence contains a dimerization motif (GXXXG). This dimerization takes place only in membranes. I had to add some SDS because the peptide aggregates in the native sample buffer.

Static light scattering is not likely to give a result because the scattering will be dominated by the lipid. Cross-linking as a function of lipid concentration is likely to be your best bet. However, if the protein aggregates without lipid (which is why you see no band in your gel) it will be very difficult to determine if it is specific dimerization or aggregation causing the cross-linking. Is the protein monomeric in 30% trifluoroethanol?

Frankly i don't know if it is monomeric in TFE... Why??

Is this your procedure:

1. Add lipid vesicles to protein

2. Run SDS gel

If so do you get a band on the gel without lipids?

I assume the protein is hydrophobic since it is a transmembrane sequence. Since it is strongly hydrophobic, the protein is probably aggregating in the absence of lipids. This is why you do not see bands at low SDS without lipids aince the protein aggregates and does not enter the gel.

With larger amounts of SDS the protein becomes soluble, however, if the dimerization is fairly weak you may only get the monomeric protein because of the small size of the SDS micelle compared to real lipid vesicles. Unlike SDS, proteins can diffuse on the surface of vesicles to find their dimerization partner. Also the physical properties of detergents can be different then vesicles, particularly in regard to membrane insertion.

The problem with lipid vesicles is you need to break the vesicle to detect the protein in the gel. A native gel with lipids will not work since the vesicles will not enter the gel. SDS will do this, however, you may lose your dimerization. Cross-linking the protein in the presence of lipids is the best bet.

TFE gives a way of stabilizing the protein independent of lipids. It therefore gives a control to see if the cross-linking is dependent on lipids.

This paper gives a procedure for dimerization in the presence of lipids. Rat IAPP is soluble in the absence of lipids. If yours is not try the running the 0% lipid lane with 30% TFE

Paper attached below

Thank you Jeffrey, i will check this paper!! In parallel, i will try the acid-urea gel dedicated for small cationic peptides!

Urea may work instead of TFE. You just needs something that the solubilizes that peptide that is not form micelles.

Another useful paper.

The file is attached:

The protocol in the paper Prof. Dubovskii recommends will probably work pretty well.

Not so surprizing. TM sequences which do dimerize in biological membranes will dimerize in SDS (glycophorin A and others) or not (erbB receptors and others). I far as I know, no one really understands why.

You may look at few papers which discuss that :

- Stanley, Ann Marie, and Karen G Fleming. 2005. “The Transmembrane Domains of ErbB Receptors Do Not Dimerize Strongly in Micelles.” Journal of Molecular Biology 347 (4) (May): 759–72.

- He, Lijuan, Andrew Hoffmann, Christopher Serrano, Kalina Hristova, and William C. Wimley. 2011. “High-Throughput Selection of Transmembrane Sequences That Enhance Receptor Tyrosine Kinase Activation.” Journal of Molecular Biology (July 12), 412(1):43-54

Also note that SDS-PAGE is alas not a method of reference for such interactions:

- Walkenhorst, William F, Mikhail Merzlyakov, Kalina Hristova, and William C. Wimley. 2009. “Polar Residues in Transmembrane Helices Can Decrease Electrophoretic Mobility in Polyacrylamide Gels Without Causing Helix Dimerization.” Biochimica Et Biophysica Acta 1788 (6) (June): 1321–31.

An alternative could be the use of some two-hybrid technique, or FRET.

Cheers

Pierre

Well I tried several gels and finally I got trimers on SDS-PAGE 20% and not dimers. I denatured the liposomes carrying this peptide at 100'C and loaded 1ug. I used 2 staining techniques (coomassie and silver nitrate) and I obtained the same results.

I would like to thank all of you for your answers...

the trimers may be dimers, just migrating anomalously. This may be due to the fact that you are comparing the migration of a hydrophobic peptide against soluble molecular weight markers . It could also be that being in a dimer causes the peptide to bind less sds molecules than expected (it binds the other monomer instead of SDS), so it migrates less , or that the bulkier shape of a dimer retards its migration (the soluble markers are linear unfolded proteins). The short sequence is also a problem, The standard rules of SDS-PAGE don't hold when the peptide is so small (i.e. the average number of SDS per amino acid is not the expected one that we observe for large proteins with a good representation of all amino acids. ).