Hi. I am currently doing western blot experiments using saliva samples.
I'm trying out different methods of extraction of the proteins in saliva. I tried extraction with ethanol. It worked fine, I also came across methods using chloroform and methanol.
Any other suggestions and recommended procedure?
Thank you!
You can please follow the links below to obtain certain valuable informations:-
1. http://scialert.net/fulltext/?doi=biotech.2008.686.693
2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797479/
3. http://www.jove.com/video/51697/collection-and-extraction-of-saliva-dna-for-next-generation-sequencing
You can please follow the links below to obtain certain valuable informations:-
1. http://scialert.net/fulltext/?doi=biotech.2008.686.693
2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797479/
3. http://www.jove.com/video/51697/collection-and-extraction-of-saliva-dna-for-next-generation-sequencing
Hi! I actually never precipitated salivary proteins, but for plasma proteins a good approach is using trichloroacetic acid (TCA) or perchloric acid (PCA), so you will denature and coagulate the proteins, followed by centrifugation (an eppendorf centrifuge will do the job since coagulated proteins will easily sediment). Thereafter, you should discard the supernatant and wash the precipitate samples (with acetone, or even ethanol) to clear off any remaining acid, again going to the centrifuge and collecting the precipitate. In principle, after these steps you should be able to suspend your proteins with a denaturing electrophoresis buffer, and load them into your SDS-PAGE system. The sample denaturing buffer should be a good dispersant, so make sure you have a suitable detergent, enough thiol reducing agents, and a chaotropic agent such as urea. A good protocol, similar to what I vaguely described is on the first reference link I appended (A nice paper, not of my authorship). The second link describes the chemistry of TCA precipitation. Be careful with TCA, it is a strong, potent acid, and in contact with skin it will cause severe burns, not to mention the risk for your eyes (therefore some people replace it for PCA, with similar experimental results. On the other hand, PCA has a slight risk of explosion...). An additional possibility would be salting proteins out with ammonium sulfate, but I really do not know whether this method will bring a more efficient retrieval of your proteins. The1st paper on the links also has a short description of it. I hope these informations will be of any help! Best wishes and good luck!
http://www.sciencedirect.com/science/article/pii/S0021967303018636
http://link.springer.com/article/10.1023/A:1026357009886
https://www.cmu.edu/ehs/chemical/Guidelines for Using Perchloric Acid.pdf
http://msds.chem.ox.ac.uk/index2.html
Thank you Sandip for the links and Julio for the elaborated answer and helpful links!
One of the papers that I coauthors has been recommended by Sandip. Some saliva samples unlike plasma samples is sometimes be viscous. You have to shake it vigourously to reduce the viscosity. Thank you
TCA method worked well for us for de-protienising and using the no protein fraction. Its quick simple and cheap.
A very helpful post! As I'm also currently doing salivary protein study. Thank you!
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