I have a few actinomycetes with me that I want to screen and separate the bioactive compounds produced out of. Can anyone please help me out with a protocol I could start with?
preliminary, easy and simple, I would suggest thin layer chromatography
Depends if you are growing in broth or on plates. If broth you will need to test both liquid and cell mass for bioactivity. I would suggest simple solvent extractions (e.g. water, methanol, ethylacetate/DCM) first followed by bioassay to determine where your activity is before you do any chromatography.
Apesar de minha experiencia ser apenas com plantas, imagino que uma secagem a 60° C seguido de trituração, maceração com etanol 80, filtragem e concentração em evaporador rotativo, poderá lhe fornecer uma extrato passível de vários testes fitoquímicos, inclusive cromatografia em camada delgada (CCD) e HPLC. O teste a ser realizado vai depender da sua disponibilidade de extrato. Se for uma quantidade pequena faz-se uma prospecção apenas por CCD. Caso gere bastante extrato pode se fazer testes de precipitação e coloração para alcaloides, terpenos, fenóis, entre outros mais específicos.
To identify them, you either do something like metabolomic study to screen the full map of metabolites and identify the differences or select a well known (or well set up) bioassay system to guid your seperation. The metabolites may be hydrophylic or hydrophobic. A general way to seperate them is to use organic solvenet to extract the crude culture (it is better to extract the culture directly without centrifuge). Then you test the extracts and water residue and confirm which one has bioactive components. It is due to the purification process for hydrophobic and hydrophylic compounds are quite different. You need to follow the bioassay results to purify your final compounds
You can select bioassay ( select the bioactivity - antifungal/ antimicrobial) based screening or do chemical profiling of extracts and fractions. Check through literature and select a method that you can manage to do in your lab ( I do not have info on what lab facilities are available to you). Have attached one paper - send me your email and will send you more papers /protocols (based on what you can do in your lab)
Although most of the replies hereabove are quite pertinent, as always in natural poducts research you should first worry about the identification of your starting organism. Then. spend some hours to gather studies published on this actinomycete or close related taxa in order to build up a chemical profile AND to look how people work with such organisms at the lab. Almost invariably methodology used and published will serve. Do not re-invent the wheel.
I agree with Alphonse's comments - the strain has to be novel to start with and cultural parameters also will influence the metabolites produced. Dereplication is important. Questions - where did your isolates come from? What is the end use targeted? Based on end use, can selective isolation protocols help in finding novel strains - Streptomyces spp are being isolated since Waksman's era - in Alphonse words we do not want to re-invent the wheel.
I am agreed with above comment but you have to develop your own protocol with some modifications using already reported methods. which method is good for secondary metabolites from action..........
this paper may help you : Identification and characterization of a Streptomyces sp. isolate exhibiting activity against multidrug-resistant coagulase-negative Staphylococci.
I personally do not know, but it may be interesting for you to contact this guy, Dr. Celso Almeida, he knows a lot about secondary metabolites from marine fungus. HEre is the link of his PhD Dissertation.
http://hss.ulb.uni-bonn.de/2011/2434/2434.htm
Good luck and enjoy!!!
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