I have to perform an ELISA assay on protein extracted from stool but I don't know which buffer is better. Daoes anyone have some experiences?
Thank you very much!
Proteins in the stool could be arising from the microbial population present in the stool, the diet, or the host tissues. Are you interested in total proteins present in the stool or a specific fraction? What is the purpose of the study?
I want to see if a human protein is present in the stool, so I'm interested in total proteins.
In this case, I suggest suspending a known weight of the stool in 5-10 volumes of 0.05M Potassium phosphate buffer pH 6.8 and vortex well. Let stand in cold for few minutes to precipitate the heavy particles. Remove the top fraction and centrifuge at low speed ~2000g for 5-10 min to remove solids and bacterial cell. Use the supernatent for your ELISA assay or for further concentration and purification by ion-exchange chromatography, gel filteration, ion chromatography or preparative electrophoresis. Good Luck
Victor Cuevas Espinoza
, in our lab we used the following protocol for protein extraction for mouse stool:"Freshly collected or frozen fecal samples were reconstituted in PBS containing 0.1% Tween 20 (100 mg/ml) and vortexed for 20 min to get a homogenous fecal suspension. These samples were then centrifuged for 10 min at 12,000 rpm and 4°C. Clear supernatants were collected and stored at −20°C until analysis. Lcn-2 levels were estimated in the supernatants..."
This protocol was found in the following article: Article Fecal Lipocalin 2, a Sensitive and Broadly Dynamic Non-Invas...
I now want to perform ELISA on supernatant from human feces. Victor Cuevas Espinoza
,which method/buffer did you end up with?
Hello Anne Mari Herfindal , do you think this technique can also used for fecal calprotectin extraction?
Adil Ömer Karakuş , yes, I would think so? I made a quick search to see what some other people have been doing. Often I see that people use some sort of extraction kit, but I could not easily find what actually the so-called "extraction buffers" of these kits contain... So... I would have tried the same protocol I think, but I would also try do to some more searches to see what other people have done... Send me a message if you want a more detailed protocol than what I posted above (like from my lab book) :)
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA