Influence on temperature on AB incubation - Optimizing immunostaining parameters (Jun/25/2008 )
Hi!
I'm trying to optimize some whole-mount immunostaining protocols - I work on small annelid worms - and was wondering about the effect of incubation temperature on both speed and specificity. I should sit and do the math, but I would also appreciate any comments on practical experience on the topic. My current protocols normally have overnight incubations in the primary ABs, and 3-5 h. incubations at room temp. I'm curious about what to expect if I move incubations to 37C.
My main aim is to enhance the signal-to-noise ratio: none of my ABs are specific for my species, and even when I get good affinity for my intended targets, I still tend to get a good deal of non-specific binding (which adds to tissue autofluorescence). I though that by twitching incubation parameters I could get there, making the parallel with PCR where higher annealing temperatures are more restrictive of lower affinity interactions. But then, I need to sit down and make the math on the kinetic equations.
I'd love to read your comments on the topic!
Cheers!
I'm trying to optimize some whole-mount immunostaining protocols - I work on small annelid worms - and was wondering about the effect of incubation temperature on both speed and specificity. I should sit and do the math, but I would also appreciate any comments on practical experience on the topic. My current protocols normally have overnight incubations in the primary ABs, and 3-5 h. incubations at room temp. I'm curious about what to expect if I move incubations to 37C.
My main aim is to enhance the signal-to-noise ratio: none of my ABs are specific for my species, and even when I get good affinity for my intended targets, I still tend to get a good deal of non-specific binding (which adds to tissue autofluorescence). I though that by twitching incubation parameters I could get there, making the parallel with PCR where higher annealing temperatures are more restrictive of lower affinity interactions. But then, I need to sit down and make the math on the kinetic equations.
I'd love to read your comments on the topic!
Cheers!
By lowering the temperature, you should be able to decrease the noise. However, after a long enough incubation, that might be nulled. Depending on the sample and the antibody specificity, I'd try an overnight incubation at a lower temperature (4C, 16C, or 20C). This tends to work for me but can vary depending on your specific requirements! The principle is simple....the higher the temperature, the more energetic the molecules will be and leads to more "interactions" in a given time. So if you have a highly non-specific antibody incubating at a high temperature, expect high background. Try to lower your temperature and incubation periods accordingly. It really is trial and error. Best of luck!! I'm sure you can figure the best option out easily.
Thanks for the reply (my Mac hard drive died the day after I posted this, so I've not been able to follow up the thread)!
So then a lower temp incubation could decrease the noise. Can I enhance SNR (signal-to-noise ratio) by raising the temp at the end of the incubation or with the following washes? It seems like unspecificly bound ABs would "let go" under those conditions, thus reducing the noise.
I'm not sure if raising the temp at the end would help your SNR too much because it will likely also raise your noise signal. You should also consider the role of various blocking agents to try and help increase your SNR during your incubations. Again, mostly a trial and error thing. No real shortcut to this type of science that I know of.
Continued best of luck!