A problem in "Solid-phase Binding between proteinX and laminin" - (Apr/15/2014 )

I'm trying to optimize an ELISA-based technique where I coat with laminin protein, block, add  proteinX and then detect for the  proteinX .

my protocol:

Solid-phase Binding between proteinX and laminin

For enzyme-linked immunosorbent assay (ELISA) analysis of binding of proteinX to immobilized laminin, wells of Costar 96-well plates (Corning) were coated overnight with 10ug/ml EHS laminin (Sigma-Aldrich) in 50 mM Na2CO3 (pH 9.6) at 4°C.

Plates were brought to room temperature and washed three times with PBS plus 0.5% Tween20 (PBST). Wells were blocked for 2 h at 37°C with PBST-2% BSA and then washed three times with PBST.

Wells were incubated for 2h at 37°C with various concentrations of recombinant proteinX in PBST(plus 2% BSA).

Following three washes with PBST, wells were incubated for 2h at 37°C with a 1:10000-diluted proteinX -specific rabbit polyclonal antiserum (this work). Plates were washed three times with PBST, and then wells were again washed three times with wells were incubated for 1h at 37°C with HRP-conjugated goat anti-rabbit IgG (Jackson, US), diluted 1:5,000in PBST-2% BSA.

After washing, 100 ul of TMB substrate solution were added to the wells in the microtiter plate. After 10 min at room temperature, the reaction was interrupted by adding 50 ul of 1M H2SO4. Absorbance was then read at 450 nm. This protocol was designed according some published papers.

But in our test, even no laminin was coated on Costar 96-well plates, protein X still can bind to plates. I don't know why.

2 possible reasons immediately come to mind (there are probably more):

1) insufficient blocking. we block with 4-5% bsa.

2) protein x may be binding to bsa.

you may want to try more and/or different blocking agent.