Help running this assay

Andrea Lopez
Andrea Lopez
Apr 29, 2024
protocol Protocol: Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate

Hello,

I recently have been trying to reproduce this protocol but adapting it to rat liver tissue and I can't seem to accurately measure GSH or GSSG concentrations. Every time I run the assay, GSH concentrations are extremely low and GSSG concentrations are extremely high (the op Read more Read less more less

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Andrea Lopez Answered May 6, 2024

Hello Vikas,

  1. We're using 100mg of tissue. We tried incorporating the BCA protein assay at the beginning of developing the protocol, but later removed it because it was affecting the results and fluidity of the procedure since another centrifugation step was necessary for this measurement. However, from these initial experiments, we would get a maximum of around 2.5mg/mL relatively consistent for each 100mg of tissue. After all the dilutions, this would result in samples containing around 25μg protein.
  2. Our buffer is sodium phosphate (0.1M)-EDTA (0.005M) buffer.
  3. For the protein assay, samples were used fresh after tissue homogenization. However, for the GSH/GSSG assay we've used both stored and fresh samples. The stored samples had almost undetectable GSH and extremely high GSSG and fresh samples increased significantly GSH but was still low compared to the literature and GSSG decreased but not that much.
  4. NEM and OPT are the only reagents that are made fresh each day the assay will be run. All other reagents are stored properly for no more than 2 weeks.


Please let me know if anything else needs clarification; thank you so much for the help!


Sincerely

Andrea L.

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Vikas Singh Author Answered May 1, 2024

Immunotoxicology Laboratory, Food Drug and Chemical Toxicology Group and Nanotherapeutics & Nanomaterial Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), 31-Vishvigyan Bhawan, India

Hi Andrea,


I have few queries before I can address your question.

  1. How much protein sample you are using for the assay?
  2. What buffer you are using for preparing your protein sample?
  3. Are you using freshly prepared protein sample for the assay or storing the sample for some time and then running the assay?
  4. Are you preparing all reagents fresh for the experiments every time?


Thanks

Vikas


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