For biodistribution ELISA is not used! Biodistribution as i understand it tracks where does the compound travel and localize within a cell or tissue, ELISA is a quantitative assay that measures amount of compound in cell/tissue/fluid lysates. Unless you are studying which cells within a tissue then you have to microdissect/dissect each cell type from a tissue and do elisa that way you can determine which cell type took up the compound within a tissue. Alternatively, live imaging if the tissues were alive (ex. PET scan) or radioactivity / nanoparticle detection post dissection (ex. mri). Subcellular fractionation followed by western blotting to determine organellar/ compartment localization. immunoHistochemistry/substrate/ emmission detection of compound from tissue slices may also tell you where it has gone and location within cells using phenotypic or special stains to identify cell types and organelles.

Thank you for your response Rabeah Al-Temaimi. Actually, In this case, I want to see whether my drug targets the tumor tissue or localizes elsewhere. And I have a specific antibody that can detect my drug in these tissue samples. So I need to know how should I prepare my sample so that I can answer how much of my protein is localized in different tissues. I am also planning to use my purified protein as the standard.

My design is as follows:

1. Collect the tissue/organ.

2. Homogenize in PBS in ice.

3. Centrifuge and discard the cells and collect the sup and proceed to ELISA.

Is this flow ok? should I consider using a lysis buffer? Although I do not expect my protein to get inside the cell.

And because in vivo live imaging needs additional steps to label my protein I have planning to do that experiment later.

Md. Masudul Haque Understood, so you want to do quantitation. From my experience I know that different organs/tissues require different homogenization buffers, for example kidney i had to use a mannitol/sucrose/Mops/ Pmsf buffer, for brain i had to use HEPES/sucrose/pmsf, but since you are not interested in the proteins but the drug I think PBS should work fine. However, for elisa a lysis buffer step is always recommended to dissociate large protein complexes that may interfere with ELISA performance (sensitivity/specificity) even if you take the supernatant of a spun homogenate. Usually, the lysis buffer includes a non-ionic detergent such as Triton X-100 or NP-40 are best, or Sodium deoxycholate (Abcam protocol worked with me for elisa protein detection). But RnDsystems have a protocol specific for tissue homogenate using PBS which should work best with your protocol, note they use freeze-thaw cycles to break proteins apart than a lysis buffer (https://www.rndsystems.com/resources/protocols/elisa-sample-preparation-collection-guide). Good luck.