Help NO SIGNAL WITH WESTERNS - (Feb/07/2007 )

Hi

I am having this strange problem where all of a sudden I have stopped seeing any signal with my westerns. .............I am dead sure of my protocol as this has worked for me for years now. its preety much routine stuff. however i did try to dilute the antibodies hoping that my problem was heavy signal. but that did not help either. sometimes i do see a signal but it disappears fast. e.g. after 30 sec exposure i see a fairly strong signal and immediately after that a 2 min exposure will give me no signal. i have tried both AMERSHAM ECL and PIERCE ECL........both have been unsuccessful. any suggestions. i am really stuck here.

-Maitreyis-

maybe you are overdiluting your reagent or it is not the strength that it is supposed to be.

try someone else's reagent (hopefully one that was used successfully recently).

-mdfenko-

I would say the opposite

I think you need to dilute your secondary and/or detection reagents further

if you read on the Pierce website, they say that overabundance of detection will result in an intense reaction that burns out quickly...

-aimikins-

QUOTE (Maitreyis @ Feb 7 2007, 07:40 PM)

in addition to mdfenko´s answer: may be your secondary Ab is down; also check your washing and Ab dilution buffer, may be wrong pH, or the 10-fold stock was used instead of the 1-fold stock (some things that happened over the years in our lab)

commercial kits have a date of expiry, and decline in work after that date especially they were used before; try to add some µl (3 per 10 ml) of H2O2 to offer more substrate in ECL reaction

-The Bearer-

thank you all for your suggestions. it was indeed high conc of secondary buffer. i diluted that and now i am good.

-Maitreyis-