Dear Alexia,

What is the detection range of your ELISA kit? Is it suitable to detect IL-1 in brain tissue homogenates? Maybe the amount of IL-1 is low in your supernatant.

Dear Maryam,

Thank you for your answer.

I use a Quantikine kit from R&D, the range is between 5 pg/mL and 2000 pg/mL. I have been able to find many studies using this kit with tissue homogenates, even though it hasn't been tested by the company itself. Reasearch groups using this kit have been able to get a signal in hippocampal tissue in similar conditions (exposure to high-fat diet), which is why I was expecting to detect IL-1 in my samples.

In case of the amount of IL-1 being too low, would there be a way to optimize the tissue processing in order to extract more protein?

Thank you

Hi Alexia,

The data from other groups, were the levels close to 5pg/ml? One obvious suggestion would be to decrease the amount of buffer used in your homogenization. Are the samples spending too much time at higher temperatures? You could try a manual homogenization to see if the sonication is not working, but my initial guess would be that the levels are too low due to the amount of buffer if other studies values are close to the LOD.

Hi Benjamin,

The levels are pretty low but from the studies I've seen, they were above 5 pg/mL. I will try to concentrate my homogenates by decreasing the amount of buffer I use.

I keep the samples on ice at all times so I don't think they were at higher temperatures for too long.

Thank you again for your help

Dear Alexia,

I do not have any experience about brain tissue.

Regarding CD4+ T cells, we had low amount of proteins for 2D-PAGE and we tried different precipitation methods in order to increase protein concentration in our samples, but they did not work. We decreased the extraction buffer volume, and it worked for us.

If a sample concentrator be available in your lab, it also might help you. Please see the below link:

http://www.labconco.com/product/refrigerated-centrivap-vacuum-concentrators/74

Best,