What buffer should I incubate HRP secondary? - (Apr/20/2007 )
Hi, I have heard that tween inhibits HRP. I use ECL plus for detection, and its protocol says to incubate 2ry HRP in TBS-T. Now I'm afraid of doing that.
Does anyone incubate the secundary in TBS? and what is the difference if I incubate in TBST
I always incubate my secondary-HRP in TBS-tween (TBS-T). Then I wash 3 times with TBS-T and twice with TBS.
It already happened that I was in hurry, and lazy to prepare TBS for the last wash, so I didn't wash a lot with TBS, but it was still working.
Maybe if you have a really light signal, it's better to wash carefully with TBS after TBS-T.
The most important inhibitor of HRP is azide. Is that what you were thinking of, perhaps?
you can use. 0.2% BSA prepaired in 50mM PBS pH-7.2-7.4. as BSA is a heavy molecule. it will help in binding of HRP conjugated secondry antibodies to the primary antibodies or what ever you are performing. Alternatively u can use TBST-20.. as it does not affect the assay....or you can use PBST-20 also... try it out..it works...as i do the same here...