ELISA - Troubleshooting - (Jan/23/2016 )

Hi,

I am planning to do an ELISA with IL-6 antibody from rat brain tissue samples. For this, I would homogenize my brain in 1 mL PBS + proteinase K. I would like to know should I dilute my samples with PBS and Proteinase K (1:5) or can I directly homogenize it in 5 mL proteinase K + PBS? I need more amount of starting material because I have to analyze 7 different cytokines. So, which method would be correct or could you suggest me another solution?

Also, I did another multi-analyte ELISA where I homogenized the rat brain tissue in 1 mL PBS + Proteinase K. I used 600 uL of that and stored the rest at -80? Can I dilute the samples again and use them for ELISA? 

Lastly, a stupid question. Is it important to determine the total protein concentration (Bradoford assay) before proceeding to ELISA?

Thanks

Hi , it is accordingly to the concentration of your tissue samples, and I suggest you do the Preliminary experiment firstly.