Can anyone provide me with the detailed procedure of how to go about with the dialysis of my LDL (L8292 SIGMA) which I oxidised by copper sulphate. It contains 5 mg of apolipoprotein as specified in its specification sheet. So what kind of dialysis bag,etc. should I use, etc. are the details that I seek to learn...
The molecular weight of LDL is about 3,000,000. If you are trying to separate it from copper sulfate by dialysis, any standard dialysis membrane will work. Avoid the very low molecular weight cutoff (MWCO) membrane because it is slow. Use something in the 10,000 to 30,000 MWCO range.
I would add that Cu(I) is quite unstable, so if you are using Cu sulfate to oxidize your LDL, you will want to use anaerobic buffer for the dialysis (closed bottle, with a purge gas like N2 slowly bubbling in) in order to prevent undesirable copper chemistry from occurring. Also be cautious of the amount of Cu sulfate you are using - too much and you absolutely will get non specific binding of Cu to your protein (and it is resistant to dialysis if bound strongly), which will also have undesirable effects on many types of experiments.
I have had very good luck using ferricyanide as an oxidizing agent, as it is removed quite cleanly. I've never used Cu sulfate before and am curious as to the protocol! :-)
My view is same as expressed by Dr. Adam B Shapiro, It should work.
Thank you Kelly for this information, I am also working on this ox-LDL work. I brought LDL and I want to oxidize it. Can you share this ferricyanide protocol for oxidize LDL.
Anandita Basu finally you oxidate your ldl? I bought LDL from sigma too, but I have problems to resuspended and oxidated the product. Can you help me with the protocol? How did you resuspend the liofilizate? Thanks.
Mc Martinez, am sorry to say but the Sigma LDL could not be dissolved though the said instructions were folowed.
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