RandomFactor

Circular Dichroism analysis of a sample is a spectroscopic technique whereby you use circularly polarised light, and then shine it through your sample, and then it gives an ellipically polarised beam at the other end... (very basic description). By analysing the ellipticity of the light at the end...you can get an idea of the helical character of the protein, or B-sheet character... It is not as direct as the NMR method, and if you were writing a paper, I don't know how it would be taken as proof of correct folding...but its something which you can use in the lab as a good indicator...it's a much faster way than NMR... I think I will wait for Yggdrasil to explain it more fully, before adding more (slight bit busy at the moment).

Yah, mostly if you dialyse with progressivly less concentrated urea, you will return back to the native protein, but the problem here is that, the chaperones acutally help with the folding... I'm not sure what you mean by "HSP70 as a part of it", but if you mean that they are functioning as they should, then maybe the technique you described would work. The problem is that you don't have any proof of it, so...the only way I can think of to get proof would be to use an NMR machine...unless you want to make X-Ray crystals, and work out the 3D structure...but lol it's better not to go there, because that would take ages. The NMR route would take you the time needed to make the samples and then probably a few hours on a basic NMR machine, and a little bit of time to compare the spectra...so it really is (in my opinion) the most time efficient way of getting direct proof that the protein has folded up properly.

Dude, The folding of the protein is highly dependant upon the nature of the amino acids used in the protein, I don't believe with the information given it would be possible for anyone to tell you whether that would refold... If you have access to an NMR machine, perhaps you can try a variety of conditions and use a 2D HSQC experiment to check if the protein looks right? You would have to make more protein which was 15-N labelled in order to do this, and would aslo have to do an HSQC on a 15N labelled "control" sample, meaning without the His Tag. These can then be compared and if the spectra near enough overlap...then "Happy Days" . Hope this helped.

I got a day's extension Thanks guys, I asked my supervisor and he told me I'm not working out % yield, but just a yield. Basically that means I should be working out mg/L not a percentage. The answers I got were correct, but they were in units of mg/L, and not % yield. Sorry about the red herring , and thanks ever so much for the help.

Hi Thanks for your reply. I had a cell culture, which contained an unknown amount of protein, and then i passed them through a "french press", basically bursting the cells open, seperated the gunk from the proteins. Then I used a TALON column (His-Tag) for the first step of purification, and then I used a gel filtration colomn for the second step. I then used a concentrator to concentrate the protein into a 600uL sample (for NMR studies). So I don't really know what the original concentration of my protein was at all, and am finding it a devil of a time trying to figure out what the yield should be. Any ideas lol?

Hi, This is my first post, I hope of many more to come, but at the moment I'm in need of a bit of help... Basically, I have purified a protein and I wanted to know its yield, but I'm not sure I'm doing the calculations right (its not an enzyme)... Here goes: The initial volume of the Flask in which I made the protein was 2 Litres. The final volume of the cuvette was 600 uL (microlitres). Concentration of Protein in cuvette was (there were 3 samples): 1) 591 uM (micromolar) 2) 456 uM 3) 2141.8 uM The molecular wieght of the protein was 7396.2 (MW). Anyone got any idea about how to go about calculating the yield? I worked out the molar quantity in the cuvette (vol x conc) and then multiplied it by 7396.6 to get the wieght of the protein (in milligrammes). I then used this figure and divided it by 2 Litres which I had in the beginning, but I'm getting some really strange figures (98%'s). Anyone know where I went wrong? Thanks very much. [bTW. I kind of need this for a write up I have to hand in on Monday, so any quick help would be REALLY appreciated lol)