I have precipitated coral slime (lyophilized, approximately 2 gm) and need to identify the proteins present inside it for preliminary lead for a project. As I can not afford to play trail and error with my sample stock, I need some established protocols for protein purification from it (coral mucus) and assurance of no chemical denaturation follow up from the protocol. Please help !!!!!!!!!
Ok, there might be many clever and better ways of doing this, but a simple and crude method to identify some proteins from your slime is to run a denaturing gel and have a mass spec service extract the proteins from the gel and analyse their sequence for you.
I would simply resuspend a small amount (0.2 to 0.5 mg/ml should be enough) and run it on an SDS-PAGE gel. Use a buffer that's suitable for marine organisms (I guess PBS might be a good starting point). Take a picture of the gel, estimate the approximate size and concentration of the proteins in your lyophilised sample from the gel, cut the bands out of the gel (use a new blade for each band or at least clean it between bands to avoid cross-contamination), label them correctly so you know what piece of gel belonged to each band on you gel and send them away for mass spec and sequence analysis.
This will work best if you only expect a few, broad bands from your sample (I'm guessing there won't be a huge number of different proteins in your slime ... at least compared to a cellular extract). However, if you expect several dozen thin bands, this might not be the best approach for you.
If you have many compounds there, a Native gel prior to the SDS gel, may help you sort things a bit more.
Thanks for suggestion guys. I have precipitated the mucus with acetone (1:4 v/v sample/solvent) and lyophilized it. The only advantage of this is to get rid from the liquid content of mucus. However the sample is still mixture of polysaccharide and protein (Brad-ford assay confirmed presence of protein). I want to go for 2D electrophoresis followed by mass spec. Obstacle here is availability of purified proteins. Can anybody suggest how to purify protein from the EPS ???????????
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