How to master HTRF phospho assays on brain samples

Tips & Guidelines
Sahra L.  
By Sahra L.    time to read 2 min

Familiar with cutting tails, tagging mice, genotyping, and finally ending up with a few mice of interest?
The cost in terms of time and effort is significant just to reach that point, before you’re even ready to carry out your phospho assay experiment!

Studying neuroscience can be a real thrill. There is so much to discover and so many questions to answer. Not only that, but recent technologies enable you to gain new insight into the field. You could be obtaining great results and getting close to publishing the paper of your life! On the other hand, maybe your phospho assay is not as strong as it should be and remains the missing piece of the puzzle.
To help you resolve this situation, Cisbio prepared a technical note to guide you towards the best possible brain sample and optimal results.

This is not a mouse. This is the mouse dedicated for your experiment.

Prepare brain lysate

Start by dissecting the animal to extract the precious brain, a very fragile organ. Pay careful attention to not crush it or damage it any way. The sample can be frozen at -80°C for storage and then thawed for your experiment.

Then lyse the tissue, centrifuge it, and collect the soluble portion.

A brain sample extracted delicately
Schematic representation of a fresh brain sample extraction with a large curved serrated

Determine optimal working protein concentration

Differences between samples are not directly detectable and can cause frustration. To avoid wasting time and energy, protein concentration must be optimized to ensure accurate results. The signal must be sufficient, while remaining within the assay’s linear range. The first step is to determine the initial protein concentration of each sample. You have three options for this. Their names are Bradford, Lowry, and Biuret.

It’s time to show your talent as a master of calibration solution sets!

A beautiful calibration solution set, designed in 2D by a great graphic designer

Set all your samples at the same concentration to normalize them. This will enable you to compare results between each sample (control sample and treated samples). Then, prepare serial dilutions of sample lysate before moving on to the assay.

Perform HTRF assay

HTRF is a reliable and accurate method for performing phospho-assays. It offers several advantages over other technologies, including measuring emissions at 620nm and 665nm, and reducing well-to-well variation. Also, it only takes one hour to run, compared to almost a full day for western blot!
Click to find out more about HTRF®.

HTRF principle demonstrated by two antibodies producing signals because of their proximity

Best practices for analyzing brain samples with HTRF®

Technical note

Download protocol