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Homemade ECL and Amersham HRP secondary - (Mar/02/2009 )

Argg..... I tried homemade ECL (Tris buffer, luminol, IPBA, H2O2).

But I get no bands at all. With the Amersham ECL kit it works.

I was wondering if the secondary antibodies we have (from Amersham) could somehow be Amersham specific? We bought a Pierce kit once and tried it, same thing - nothing.

What's up..? Any help/advice? Wish we had some none Amersham secondary I could try out.

-MKR-

Nevermind, I tried three different secondary antibodies. I used the Homemade ECL mix and put 1mL per tube and then added 1uL of each secondary-HRP ab. All tubes luminesced.

I'm really stumped as to why I'm not seeing any bands on my blots. What the heck is in that Amersham mix that makes it work when the Pierce and our homemade show absolutely nothing?

I was really hoping either not to see anything or at least not to see anything in our Amersham secondary tubes. Now I am really stumped?

The only thing I can think of is that the Amersham ECL kit really is very sensitive, and the Pierce and homemade stuff just can't detect anything (but I really feel like we're not looking at super low abundance stuff - I load ~20ug protein per lane, and even my actins aren't showing any bands)?

Any ideas?

-MKR-