Hello there,
I have prepared 10-55% sucrose gradients containing 1mM Chloramphenicol and run them on an Isco sucrose gradient fractionation system, but the absorption is too high to record on the system's chart, even for just the gradient not containing any samples. Have you encountered this before? Is there a workaround for this?
Are you using the correct filter for the wavelength you want to use? Also, is the detector working correctly? In other words, is there something making it think there is absorbance?
Hello Jack,
Thank you for your response, I think the filter (254nm) is just fine when we use it in other conditions, because we routinely perform sucrose gradient fractionation to observe polysome profiles - and they work perfect when Chloramphenicol is not present in the gradient, for eg. in the presence of Cycloheximide (for mammalian cell lines).
Chloramphenicol has a lambda max of 278nm - so I hope the supplied filter does not have a range broad enough to pick this up.
There are 2 ways to reduce the absorbance measurement: change the wavelength to one at which the absorber has a smaller extinction coefficient and reduce the pathlength in the detection cell.
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