Stjepan Krešimir Kračun

Brief protocol.

1mg of pure protein was in carbonate buffer pH 9.0

1mg of HRP was diluted in ddH2O.

A 100mM Sodium Periodate was prepared and 100uL of it added to HRP (100ul in 1ml of HRP) - 20 min incubation in dark. Then the residual NaIO4 was removed with Sephadex 25.

The mixture of NaIO4 was added to 1mg of my protein. And incubated for 2 hours in dark.

4mg of Sodium Borohydride was diluted in 1ml of ddH2O.

400ul of Borohydride was added to the incubated mixture of protein+HRP+NaIO4 under agitation and incubated for 2 hours.

The final mixture was desalted again using Sephadex 25.

Unconjugated protein distinguishes positive and negative human sera for Shipylis in an protein+serum+antiHuman ELISA.

Conjugated protein makes the signals of positive and negative sera have the same values.

The ELISA I am developing is as follows (no possibility of changing the strategy)

Solid-phase: Unconjugated protein.

Secondary phase: a mixture of conjugated protein + serum

TMB then stop.

Stjepan Krešimir Kračun

The only change you purpose is to instead of removing the residual NaIO4 by Sephadex 25, quench with Ethylene Glycol? (if so, in what concentration?)

Won't it the be same, removing residual NaIO4 or quenching it?

Thank you and best regards

Stjepan Krešimir Kračun

So, as I understood, after eliminating the residual unbound NaIO4 with sephadex 25.

The bound NaIO4 to HRP still needs to be quenched. Then I add the protein?

Thanks again

Stjepan Krešimir Kračun

I will read ther articles and the link.

But I found where was my error. Actually it was in typing you the protocol.

1 - I add NaIO4 to activate the HRP

2- Use Sephadex to remove NaIO4

3- Then I add activated HRP to the Protein.

4- Finally I add NaBH4 to close the Schiff bases.

And Still have my ELISA signals going completely crazy.

Stjepan Krešimir Kračun

HRP activation for 20 min in the dark

HRP - protein 120 min in the dark

HRP/Protein - NaBH4 - 120 min in presence of light

Thank you