I found Antibody against Glutamate is available...but is it possible to perform WB for a single amino acid?
no, it isn't. it would not stay easily in the gel and it won't bind to any membrane. for single aminoacids, chromatografic methods are used. but for quantifications, it is better to go to liquid chromatorgraphy; elisa may work, but will be very noisy.
Sounded odd to me at first, searched it, seems legitimate
http://journals.lww.com/neuroreport/Abstract/1999/11260/Co_localization_of_glutamate,_choline.10.aspx
http://onlinelibrary.wiley.com/doi/10.1046/j.0953-816X.2000.01328.x/abstract?deniedAccessCustomisedMessage=&userIsAuthenticated=false
But not as WB, rather as dot blots
no, it isn't. it would not stay easily in the gel and it won't bind to any membrane. for single aminoacids, chromatografic methods are used. but for quantifications, it is better to go to liquid chromatorgraphy; elisa may work, but will be very noisy.
Hi! If you are looking for a reliable and cost-effective method for glutamate determination in liquids, I could recommend the Amplex Red Glutamate Kit (available from Molecular Probes but the individual components can be bought from Sigma or other sources). It is remarkably sensitive, robust and easy to handle. All you need is a microplate (or cuvette) reader capable to read fluorescent output (preferable) or colorimetry. For Glutamate in tissue (brain) I have also used an antibody (Chemicon), but quantitative conclusions are difficult. ELISAs are available but -to my knowledge- are more expensive and less sensitive than the abovementioned Amplex Red Kit.
I have lysates extracted from mouse brain... then what should I do to determine the level of Glutamate?
In lysates the Amplex Red should work. Due to its sensitivity a high dilution factor of the tissue lysate is possible. Centrifugation and/or filtration steps may be necessary to clear the lysate from interfering debris. Please note, that in tissue lysates you will detect intra-AND extracellular glutamate. I assume that you are more interested in extracellular glutamate. To determine this, you would have had to perform microdialysis. I usually measure (extracellular) glutamate in cell culture supernatant. You should check, wether this combined measure of EC and IC glutamate makes any sense.
well, first you should ask why you want to measure glutamate in brain homogenates. If you want to measure neurotransmitter glutamate, your measure is basically meaningless. If you want to measure metabolic glutamate, you should pay attention to tissue preparation and standard homogeneates may be not good.
i want to measure the neurotransmitter glutamate? tell me about the ways to measure it.
you want to measure glutamatergic transmission in whole brain, in some areas, in specific cells, at specific synapse.... and in which model?
I was reading the thread and impressed by the inputs. If any one still following this thread, could you please tell me how could one measure the level of extracellular Glutamate released in Hippocampal cells? Thanks
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