Hello,
I have about 1 mL of snake venom. what is the best suggestion for analysis of its ingredients?
Hi, depend of what you want to do, I think that HPLC is the best ways to find out what is the component of venoms depending of size, charge or hydrophobicity.
we want to know the types of enzymes and peptides (as well as their ratio).
Hi Dr Farzad Seidi ,
If you want to know the proteinaceous compound of some snake venom, i suggest you make a venomic study. You could to make 2D Electrophoresis and after, sequencing by mass spectrometry.
Best regards,
Hi Dr.
What kind of appliances do you have at your disposal? I am asking about HPLC/UPLC, MS.
The most usual way would be a RP-HPLC followed by MS/MS.
we have Electrophoresis, HPLC and MS. But we don't have MS/MS, HPLC/UPLC,...
If you have only MS, may be a good idea to separate fractions using HPLC, then fingerprint them with trypsin prior to MS analysis. The fragment´s masses may give an idea of what you have in your venom.
I think Alexandre is right, fractionning with HPLC will be the best but before going to MS analysis with trypsin I will suggest you to test for enzyme activities on your fraction. You may have an idea about what kind of enzyme you're looking for !!! Hope it will help. Good luck
The first points before any suggestions are:
1) from which snake was obtained the venom? (is not the same to study an elapidic venom or a viperid venom or a colubrid venom).
2) What do you want to know about your sample (biochemistry, components, toxicity, etc.).
3) In which state is the sample (liquid and frozen, lyophylized, vacuum dissecated, in glicerol, etc.).
After to know this, a rational and useful advice could be suggested.
The best regards,
Adolfo
Hello,
(1) The venom has extracted from a viper snake.
(2) we want to know the following information: LD50, enzymes and their activites (such as phopholipase A, L-amino oxidase, .......), protein content,
(3) One sample is in the vacuum dissecated form and also there is a sample in the lyophylized form.
OK. Now is more clear.
Considering a viper venom.
I you want to make separation of fractions avoid to use HPLC if you want to test the toxic activities since several enzimes as metalloproteinases can be inactivated by the solvents.
The same for liquid chromatography, you should choose the buffers and pH carefully to avoid the inactivation of enzimes, like for example the mentioned metalloproteinases. You will have a range of proteins from around 5 to 80 kDa with a wide range of pi. The main components possibly will be metalloproteinases, phospholipases and proteases of serine, with several groups of enzymes and toxins which will vary regardint the genus and specie.
If it is a viper venom the toxic activities that could be studied (among several) are the LD50 (possibly 18-22g mice intraperitoneal could be a good choice). The hemorrhagic activity as well as the coagulant on plasma and fibrinogen, the defibrinogenating activity could be useful. Among the enzimatic activities the phospholipase and proteolytic on gelatin (or other substrates related with the extracellular matrix) could give you some information on the toxicity of the venom. You have all these techniques in Theakston, R.D.G. y Reid, H.A. Development of simple standard assay procedures for the characterization of snake venoms. Bulletin of the World Health Organization 61, 949-956, 1983 or in World Health Organization. WHO Guidelines for the Production Control and Regulation of Snake Antivenom Immunoglobulins. 2010, 110. http://www.who.int/bloodproducts/snake_antivenoms/SnakeAntivenomGuideline.pdf.
There are not big differences in toxic activities in the venom lyophylized or vacuum dried. Of course can be some differences in some enzymatic activities, but the toxic activites are quite stable in these conditions.
The best regards,
Adoflo
Needs to analyze various protein, amino-acids, and enzymes. Immuno-assay techniques may be useful.
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