Conjugated microspheres dilution question - Troubles in diluting streptavidin conjugated microsphere (Aug/27/2010 )
I am PhD student of Tokyo University working with Mucosal immunity ( vaccine strategies, precisely speaking). A Couple weeks ago I have obtained streptavidin conjugated microspheres (Fluoresbrite Yellow Green, polysciences) in order to conjugate with my biotinylated mAb for orally delivering them.
My problem is, at data sheet, there is no clear information ( at least for me) about dilution of microspheres. They just say the particle concentration is 1.25% and you must resuspend it (after wash) at ( at least) 5x 10'8 particles/ ml concentration.
I am wondering if somebody here have used this particle before, if so, Do you have any idea how can I dilute ( define the right dilution) this particle at 5x10'8 or 10'12 concentration using only the information provided for them ( particle concentration : 1.25% )?
I already e-mailed the company, but 2 weeks have passed and I had no answer.
Please, I know maybe my question looks like so naive question, but if you have used this product before, we will help me a lot providing me that information.
Thank you in advance
PS.: Here is the product data sheet: http://www.polysciences.com/SiteData/docs/TDS%20616/cbd811c649141ed4402d71db2fd159ff/TDS%20616.pdf
From their specification sheet: 5 x 108 particles/ml and 15-40μg of biotinylated antibody/mg of microspheres.
It would be wise to check similar protocols from other vendors such as: Seradyn/Thermofisher; Invitrogen/IDC, Varian/Polymer Labs. Many of these are on-line under 'technical notes' 'procedures' or 'applcations' on their respective web sites.
You should probably start with holding the mass of latex constant and varying the biotinlyated-protein. Finally, you would vary the volume of protein-lx conjugate in your assay to determine the optimum.
Here is another article: http://www.bangslabs.com/files/bangs/docs/pdf/PDS_215_218_219.pdf Note: Polysciences and Bangs Labs are the same company...