2 replies to this topic

[eilidhfiona]

Does anyone know of a good anti-goat-HRP conjugate secondary antibody?  The one that we use gives very high background on western blots...  Thanks.

[aleruiz]

The secondary antibody is not the only one factor in a high background. The blocking agent and the specificty of the primary antibody are crucial factors. The exposure time in the autoradigraphy is imporant too- too much time could be bad. I use several secondary antibodies from Santacruz and give good results. Check your conditions in the assay and don´t trust only in the secondary.

Good luck.

[serglom]

eilidhfiona, on Nov 10 2004, 07:35 AM, said:

Does anyone know of a good anti-goat-HRP conjugate secondary antibody?  The one that we use gives very high background on western blots...  Thanks.

You may try with:

- Increase secundary dilution (not less than 1:2000)
- Change blocking conditions (increase time, increase blocking agent you use,...)
- Time exposure (take images at different exposure times)
- Dilute primary antibody
- Charge less protein (You can try differents quantities)
- Wash conciously the membrane (6 x 5 min in agitation is good) between incubation with antobodies
- Use blocking reagent with the antibodies (non-fat dry milk, perferct-block or BSA)