Hello,

Theoretically, in a SDS-PAGE electrophoresis  proteins are fully denatured (you can be sure it completely if previously heat up the samples in loading buffer: 100ºC for 15 min), and the electrophoretic mobility depends only on the molecular weight (except when cofactors are covalently bound , which may sligthly affect the mobility).

Good luck.

No, Not at all, SDS-PAGE cann't be used for assessing extent of protein denaturation, CD is the method to measure it.

As Carlos and Rakesh said, SDS-PAGE needs fully denaturation of protein and this can not be used for comparison.

Again as Rakesh said, circular dichroism (CD) is the best method for evaluating the secondary structure of the protein. The 2nd structure is fully destroyed in denaturation of protein.

Hi, SDS-PAGE cannot be used as a tool for studying the extent of protein denaturation. Apart form CD studies (which is the best method), you can also go for fluorescence study and/or HPLC analysis and make a comparison of your isolated protein before and after denaturation. 

SDS-PAGE is of limited value in studying protein denaturation.  By itself, SDS-PAGE cannot assess protein folding very well given that by the nature of the method, proteins are denatured by the SDS, which can be faciliated as mentioned above by heating the sample.  However, if the protein is soluble, you can test the the susceptibility of your protein to chemical or heat denaturation and then measure the amount of folded protein remaining by SDS-PAGE.

For example, I recently measured the thermal denaturation isotherm of a protein by incubating different aliquots at increasing temperatures.  Then I centrifuged the tubes and collected only the supernatants.  I ran the supernatants in SDS-PAGE to see how much protein remained in solution; this was the remaining properly folded protein, and the denatured protein aggregated and pelleted in the centrifuge.  My isotherms were comparable to what was previously reported by circular dichroism.  You can also do this with different concentrations of chemical denaturant such as urea or guanidine hydrochloride, but these may affect how your protein runs in SDS-PAGE and may have to be removed.

If you're wondering how much of a protein sample is properly folded, CD is definitely the best method, especially if you already have a CD spectrum of properly folded protein to compare to.

Really these responses have been helpful, however can I get a link to a suitable reference on both methods in reference to protein folding and it's molecular weight.

Dear Shih

Your point of view is very valuable.

What is your idea about heat stable protein?

Is there any simple method such as you mentioned above?

Masood, the protocol is actually very simple and inexpensive.  An old paper which uses such an approach is here, specifically in Figure 4:

Pedersen KE et al. Plasminogen activator inhibitor-1 polymers, induced by inactivating amphipathic organochemical ligands.  Biochem J. 2003 Jun 15;372(Pt 3):747-55.

http://www.biochemj.org/bj/372/0747/3720747.pdf

You just need to have several water baths or heat blocks going at the same time for a range of temperatures.  Depending on the temperature range you're studying, a gradient PCR machine might work.  You also have to be careful when you pipette the supernatant from the pellet after centrifugation.  This gives you very blunt information about the overall protein stability profile; however, as I said, it's an experiment that can be performed in many lab settings.