I bought a lipid peroxidation kit from Sigma and I want to get kits to measure nitrates, superoxide dismutase, catalase, and glutathione. Each one of those kits comes with a different lysis buffer. But to use all those different lysis buffers I would need a lot of samples. What I am wondering is, there is any solution that I could homogenize my samples with that should be compatible with all those tests?
Hi Danielle,
Answering this question becomes challenging without insight into the specific kits you intend to use and the underlying measurement principle. It's crucial to avoid any interactions with your analytes, like catalase inhibition, which may impact downstream measurements. Additionally, ensure that the sample lysis process doesn't impede subsequent reactions used to evaluate the concentration or activity of the analyte. My experience with standard lysis buffers didn't result in interference with the mentioned measurements, but it's essential to consider the magnitude of the effect you aim to detect and the nature of the procedure or reaction you're employing.
In general, relying solely on kits might not be ideal. While they offer convenience, validating their suitability for your samples and experimental setup often requires significant investment in validation and control conditions. Sometimes you'd need to buy several kits ensuring you have enough reagents for all the important control conditions. Producers may be reluctant to disclose specific reagent contents, and inquiries about potential interferences may lead to disappointing responses, as such tests might not have been conducted. Fortunately, numerous simple and cost-effective biochemical methods are available for the tests you wish to perform, which you can easily establish in your lab with minimal expenditure on reagents. Though it may require some time, having a method you can manipulate indefinitely is invaluable for ensuring everything functions as expected and eliminating concerns about potential interferences.
Another important thing to consider in your case is what you plan to use the samples for, apart from the tests you mentioned. If you don't need to inhibit specific reactions (e.g., phosphatase or protease inhibition for downstream WB) and plan to run everything in a short time window, why not opt for a simple buffer like PBS?
Hope this helps a bit!
jan
Dear Danielle.
For several decades now, I have regularly measured the activities of a large number of antioxidant enzymes and several substrates, including the reduced and oxidized forms of glutathione, on various types of cells or tissues.
I haven't used chemical lysis for a long time. I prefer mechanical lysis, using ultrasonic disruption. I proceed on samples that are still frozen, onto which I pour a buffer, such as PBS or other, without any different component. I deliver the ultrasound to the still-frozen sample pellets. I proceed over 20 to 30 seconds, pulsing 10% of the cycle. The energy released should not be too strong. The tube in which I sonicate is immersed in ice to avoid heating the sample. Samples prepared this way are compatible with the many assays I perform. Fast operation ensures safety, and dosing is done in a flash.
For glutathione, the sample must undergo acid treatment immediately after homogenization to avoid further degradation, which is very rapid. To this end, an aliquot of the homogenate is poured into a 2% PCA solution.
I hope this will help you in your research.
Best regards.
Marc
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