ELISA Kits for proteins in brain homogenates - (Oct/05/2011 )
I need to assess some proteins in mouse brain homogenates. I never did ELISA before, so I would like to ask for some tips for purchase ELISA Kits. Moreover I think that our candidate proteins are not very common to assess (BDNF - this one is quite common, IGF-II, Sonic hedgehog). I've already found few kits for them, but usually they are recommended only for cell culture suspension or plasma and other liquids. Can you please give me an advice where and how to look for proper Kit for less common proteins? Or if somebody has an experience with brain homogenates I would be happy for any advice!
Thank you in advance.
You can certainly try the kits with your homogenates. Make sure the fluids are filtered. The issues you may have to address are matrix effects. If you have the protein purified create a dose response curve in the buffer used to create the homogenate. Test these standards/calibrators first in parallel with the kit calibrators/standards. Your curve should lie on top of the other curve or be in parallel with it. If it lies on top that is a good start, if it is in parallel you can either use your calibrators or the kit calibrators that you reassign your values to.
Your samples will have other proteins and, of course, other issues can appear. Cross reactivity, interference etc.
Thank you for your reply. All right, I will try, the problem is that the Kit for Sonic hedgehog is very expensive so I hope I won't waste all our money You mean that I should centrifuge the homogenates and then filter the supernatant? (which filter please?) There is usually only centrifugation step in protocols I've seen.. I am also wondering what dilution of samples is best to start with? We are going to use only mouse cerebellum and donť know how much protein will be there..
You don't want to add any cells/debris to the wells of the kit; a 0.2 um should let your proteins pass and remove debris. Just do 2-3 points first 0 mid high of the kit AND 3 ten fold dilutions of your protein/buffer (ie 1:10, 1:100, 1:1000 plus buffer only) so that is 7 points leaving you 89 wells.
Ok, thanks again!
You should also do a OD 240 to get a general idea as to the total protein concentration of your filtrate...you may want to have some idea as to your protein conc v. the Total
Let us know the kits you are intending to try, some are good, some are so/so, some simply don't work. The key to this project is to identify an appropriate solubilization solution to prepare your brain homogenate, and it may vary from molecule to molecule.
Western blot type people will advise you towards denaturing buffers, but remember that these will also denature the assay components. As you are breaking up the cells of the brain tissue, you will be releasing a whole load of proteases, so you will need to include a broad spectrum protease inhibitor and EDTA within the homogenization solution. A good PI cocktail to try is sigma P8340 you could also try their cell lysis kit (MCL1)
Beware of filtration, because you may throw away your analyte in the retentate if the solubilization is not optimal. You may want to consider a solid phase extraction (SPE) step which will result in a much simpler matrix to work with in the ELISA. What ever you decide for solubilization/filtration or extraction, determine the extraction efficiency by spiking a known amont of analyte into the homogenate, and ensure that you have reasonable and consistent extraction efficiency which will allow you to express results per mg of wet weight tissue (calculating for dilution and extraction efficiency).
Protein content analysis at 240 won't work, you would use 280 or BCA but your homogenization buffer itself is likely to have protein in it, making protein content assessment difficult hence the standardization based on starting tissue weight.