how do u determine actual concentration from absorbance readings? - using tissue lysates for ELISA sample (Aug/06/2008 )

Hello,

The BDNF concentrations I obtained from the ELISA plate reader cannot be the actual concentrations of BDNF in my lysate samples because the Bradford Assay gave me different concentrations of total protein to begin with. How do I "normalize" or take into account the original difference in concentration of my protein lysates to show the ACTUAL concentration of BDNF my samples in ELISA???


Here's some more info on what I did:
-> I used a Chemicon BDNF Sandwich ELISA Kit
-> My standards/standard curve for both Bradford and the ELISA plate worked just fine
-> Another way of phrasing my question is : Is Total Protein concentration supposed to be proportional to ligand concentration in ELISA???


I hope my question makes sense... I tried my best..

-frustrated lab tech

Can you post your results?

If your BDNF is pure in your sample with no other proteins present. then the Bradford and Elisa will have similar but not exactly the same concentrations.

You are doing two different methods. If they were two different elisas then the results would be much similar.

So, if the sample is pure run both methods, two dose response curves. Run your unknowns (over the linear portion of the curve). Plot a methods comparison, from your slope and intercept you will be able to directly compare results between the two methodologies.


Hope this helps.

hi dear,

take your samples run same volumes on SDS-PAGE,
stain with commassie.
make densitometry of all ur samples.
normalize them to a value and nomalize the total protein concentration.
this one of the best ways i worked out when i had problem with bradford and BCA methods of estimation.

gud luk