Biofluid is infected by some microorganism, and i want to extract the total protein present in biofluid as well the infected microorganism's protein.
From the topics you flagged I suppose you want to do this for western-blot purpose. I find the in Thorner buffer (8 M urea, 5% sodium dodecyl sulfate SDS, 40 mM Tris [pH 6.8], 0.1 mM EDTA, 04 mg of bromophenol blue per ml, 1% β-mercaptoethanol) as the best for your biofluid. When you will boil it in the mentioned conditions, all the hydrophobic proteins will become soluble. I cant imagine microorganism will not be destroyed under such a harsh conditions. Only you should be careful is to wash off the urea from the slots after the bromophenol will enter the stacking gel.
Thank You Jan
My work is basically meant for biomarker discovery in the patient's biofluid which can be invasively collected (like Saliva). My concern is that I am trying to extract all the protein (including the host as well as the infected organism's protein) present in that body fluid . So that I can search a marker protein for that disease.
Is that buffer help me to extract the hole protein from the biofluid for mass spectroscopic analysis.
Sorry for this delay in answer, due to my holidays. From saliva you would easily extract the proteins for MS with the Thorner buffer. You can precipitate them prior the trypsin digestion. The precipitation is strongly suggested due to urea presence, will disturb the digestion. By this step you will also throw away the polymers present in saliva, can disturb the MS analysis. I don't have the experience with protein precipitation from saturated urea. Thus I suggest you to dilute your sample with water 10x before and do the cold-acetone precipitation (suggested by the people from my MS lab). Please remember that for MS rather the quality than quantity is important, huge pellets are not recommended. The sample corresponding to 2-10 microliters of your saliva sample should be reasonable for initial MS. Keep fingers cross:)
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