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I am getting significant signal with this antibody diluted 1:1000 in mouse 3T3L1 (preadipocytes) without the addition of any primary antibody. I fix these cells in 4% PFA for 15 minutes and wash 3Xs 10 minutes. Further, I have blocked using 5% donkey serum with 1% BSA, 10% donkey serum, and 10% horse serum for 1 hour at room temperature all with similar results. Any other suggestions?
Asked on Apr 04 2013
Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab150129. I would also appreciate if you can confirm some further details: Do you use a detergent such as Tween 20 in your washing buffer? Have you attempted to increase the number and duration of washes after antibody incubation? If so, did this have affect on the background? Have you attempted to further dilute the antibody to 1/1500, 1/2000 or greater? This antibody has not been pre-absorbed against mouse IgG, as such we cannot guarantee that we will be able to eliminate this reactive thought we hope that these suggestions will help reduce this. Abcam does offer a number of AlexaFluor 488 antibodies which have been pre-absorbed to ensure low species cross-reactivity including ab150133 which has been pre-absorbed against chicken, human, mouse, pig, rabbit and rat. More details about this product may be found at: https://www.abcam.com/donkey-polyclonal-secondary-antibody-to-goat-igg-hl-alexa-fluorreg-488-pre-adsorbed-ab150133.html
Answered on Apr 04 2013