Hello. I hope someone can help me or give me an idea on how to prepare HEPES buffer with Chelex-100 using the statement I have read on a published journal.
Enyzme (500nM) was incubated with aspergillomarasmine A or AMA (500 uM) for 10 minutes. 20 uL of the above was diluted with 180 uL nitrocefin or FAPGG substrate for the following concentrations: Enzymes (50nM), FAPGG (50uM)/nitrocefin (20 uM), AMA (50 uM). Buffer (50mM HEPES pH 7.5, 200mM NaCl) was stirred overnight with 2g/100mL Chelex-100. Assays were read in 96-well microplate format at 490 nm using a Spectramax reader at 37 degrees Celsius.
Hi Alecks Megxel Serrano Abordo . I am guessing that Chelex-100, a chelating resin, is used there to get rid of any polyvalent metal ions in the HEPES buffer. It shouldn't be needed if you are using good quality HEPES and di water.
I agree with @Steingimur Stefansson. If you nonetheless need to go ahead with the Chelex treatment, ther is nothing more that I can add besides what is already described in the recipe: add 2g/100 ml Chelex to the buffer, stir overnight, preferably by shaking and avoiding stirring bars that may damage the resin beads, decant the beads and take the supernatant.
It would probably be a good idea to 0.2-micron filter or centrifuge the buffer after the Chelex treatment to remove any particles that may have broken off the beads.
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