Hi all,
I would like an advice. I am detecting a xenobiotic protein I injected into mice in mouse organs: liver, spleen, blood, brain, etc. I am planning to firstly collect and snap-freeze organs and then plan to thaw them, weight an exact amount of each organ I need (e.g. 10 ug) and run a detection using a commercially-available kit.
I want to know if my method is appropriate or whether I shall fistly weigh how much tissue I need, then freeze that fragment and then run deteciton of the protein in it? I want to reduce the variation between samples by making them the same weight. Though, there is a different number of cells in each organ, so how should I approach it?
Thanks,
Maria
Hi Maria!
Did you try to attach a fluorescence indicator to the xenobiotic protein before injecting it into mice? If yes, you can use the IVIS method to detect and measure the distribution of your protein in the whole mouse body, in the organs you want. This method is used for live animals.
Hello Maria,
I follow the protocol given below.
Whenever I need tumor tissue for IHC, I excise, weigh the tumor tissue and section a small piece of tumor and fix it in paraformaldehyde for hematoxylin and eosin staining and immunohistochemistry. I immediately snap-freeze the remaining tissue in liquid nitrogen and store it at −80°C for other analysis.
So, I suppose you should first weigh how much tissue you require, then snap-freeze that fragment as well as the remaining part of the organ in liquid nitrogen and store it at −80°C for proteomic analysis. Collecting, snap-freezing the organs and then planning to thaw them to weigh an exact amount of each organ would not be an appropriate method according to me.
Best.
Protein Quantification:
- Colorimetric Assays: Use methods like the Bradford assay, BCA assay, or Lowry assay to determine protein concentration by measuring color intensity.
- Spectrophotometric Methods: Measure absorbance at 280 nm to estimate protein concentration based on the aromatic amino acid content.
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