While doing GSH on rat mitochondria, I have calculated protein concentration by Bradford's assay (1.35mg in 2ml buffer). In case of GSH assay I got concentration of 0.23mM (GSH) in mitochondria. So, please suggest how to find GSH present in mitochondrial protein in umol/gm?
What volume of sample did you use for the assay? You have 0.675 μg/μL protein in your sample so multiple by the amount used for the assay. GSH has a GFM of 307.32 g/mol so you could also convert it into μg/g.
Thank u so much for your answer. I have done Bradford's assay after incubation with a suitable buffer so the volume becomes 2ml. and got the value of 1.36mg protein / 2 ml of buffer. Further, I have taken 100ul of mitochondrial sample to perform a GSH assay, and the value of 0.23mM/1.35mg I found.
So can you provide me a sample paper that how you have calculated 0.675ug/ul ?? or elaborate on it, please?
Additionally, how you have found a GFM (stands for) of 307.32g/mol?
Thanking You in advance.
So you diluted your sample with x amount of buffer and then did the Bradford assay? Was the 100μl used for the GSH assay with or without the added buffer?
If you used the same solution as you used for the Bradford assay you divide the total protein in the sample by the volume (1.35mg/2000μl) and then multiply by the amount used for the GSH assay (100μl). If the sample used for the GSH assay was not diluted with buffer you will need to divide by the dilution (sample vol/(sample+buffer)) first.
GFM stands for gram formula mass and can be found on most chemical data bases (eg. Chemspider).
I have used the same which I have used for Bradford. I.e. diluted sample.
At last after calculation....
I'm getting 230um(GSH)/0.0675ug(protein)
230um/0.0675ug is this u mean?
Tysm for response
Firstly 1.35mg/2000 is 0.000675mg/μl or 0.675μg/μl. As you used 100μl of that solution for the GSH assay you had 67.5μg protein in your GSH assay. Therefore with 230μM GST/67.5μg protein you have 3.407μM/μg protein.
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