Hi,
im looking at PAL activity in blueberry samples. I use L-Phenylalanine as a substrate and absorbance at 290nm. The absorbance values of my blanks are higher than 1, even my milli pore water is above 1. I diluted buffers, substrate... even remade everything. i am out of options.
my protocol is as follow:
acetone powdered tissue extracted with borate buffer pH 8.8 and PVPP.
75ul sample extract
150ul (l-phenylalanine 30uM and borate buffer 30mM)
after 30mins rxn stopped with 4N HCl.
absorbance at 290nm
my latest results are very close to each other (0.8-0.999) my blanks are above 1.
any ideas to what might be the problem?
If Milli-Q water is giving an absorbance greater than 1, you have a problem with your spectrophotometer or your use of it. Are you using a quartz cuvette? Glass cuvettes absorb UV light.
Im also thinking it might be the spec. I use microplates. I will try using different ones. These ones were brand new.
Standard polystyrene microplates are opaque at 290 nm. You require UV-transparent microplates.
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