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Non-specific staining with secondary... - Scott are u there?? (Jun/25/2005 )

Ok this is the thing...

I am using the Alexa flour 633 goat anti-mouse secondary for staining one of my proteins... Unfortunately in the well that I add just the secondary alone (to check for non-specific binding) i get a signal. This is however not the case with the alexa flour 488 goat anti-rabbit.

The folks at molecular probes tell me that I need to titrate the product. However, even at a dilution of 1:900 ( i normaly use 1:400) there is non-specific binding. I adjusted tha amplitude gain on the microscope such that the signal of the secondary alone goes away. Then when I screen the cells that have the primary + secondary together and scan them using the same amplitude gain I get no signal i.e the signal is extremely weak.

To rule out the possibility of the the primary being the culprit...I used two different antibodies for two diff proteins and it gives me the same result. I dont know what the problem is...

All the help will be appreciated...I hope I have stated my problem clearly.


Hi Pria,

I have often had problems with co-localisation in the far end of the spectrum (far red/blue) with background. I find that the excitation and emmision spectra are closer resulting in a direct reading from the laser itself. Therefore I have previously tried a few things.

1/ Worst primary detection (least abundant) I will use with Alexa-488, in my hands this is the most sensitive secondary. Conversely most abundant I'll use in the Alexa-633 as this will give a stronger signal and therefore widen the window between specific and background staining.

2/ Drop the laser power. This should have the effect of minimising direct detection via the laser, however, you may have to increase the gain to get your image.

3/ If the problem is with the secondary antibody can you increase your non-specific blocking. I always incubate my antibodies in the presence of BSA. It may also be worth using some serum of the tissue/cell source to use in blocking.

4/ Finally the only other thing I can think of is check your filters to ensure you are using the correct filters. I also find that the red (Alexa 594) is more sensitive, but obviously its use is dependant on your laser.

Hope this helps, I haven't done this stuff for a while so are a little rusty.




Hey Scott,
Thanks a lot....
ok this is the problem..I am already using alexa 488 anti-goat for my primary protein ie. the most abundant and i do not want to change it. I am using the far red for the secondary protein (s). I agree that 488 is also the most sensitive in my hands too.

I am already using %% goat serum for 1 h toblock. These are human breast cancer cells. I specificaly use GS as I read some place that if my secondaries are derived from goat (which is the case) I should be using GS for the blocking. U think the BSA will work in this scenario?? if so what % should I use??

Also I add my primaries and sec in 2.5% GS.Do I need to chnage this.

I use the phalloidin 546 for my actin staining. This complicates my use of 594.

I am using a zeiss510 laser scanning mscope which covers the entire spectrum so I am +ve its not the prob with the filter.

Thanks a lot for ur reply since It definitely helps me.


Hi Pria,

I stumped, I'd be interested to see what others think.

Again the only three things I can think of is to switch the colours ie. use an Alexa488 anti-mouse and an Alexa633 anti-rabbit.


As the techies from Molecular Probes suggest dilute out your secondary antibody, you could even try a ten fold dilution (ie. 1 in 4000). Hopefully you would get to the point where there is little or no excess antibody sticking non-specifically.


Drop the power of your laser. Because excitation spectra have a distribution the more intense your excitation then the larger proportion will be directily detected by your filters.

Additionally I guess you could try and find a different clone of secondary antibody conjugated with something in the far-red. Maybe that clone of anti-mouse is not fantastic.

If I think of anything else I'll make sure I post it. Let me know how it goes.

Good Luck



thanks scott...


-- Are you using primary cells grown on slides or coverslips or tissue sections?
-- How did you fix your cells?
-- I use the goat anti-mouse alexa conjugates and find that you can use them 1:2000 (which for my vials is 1ug/ml). You can probably even use them 1:4000. Molecular Probes sells their secondaries at 2-4 times the concentration of many other companies. I never go by dilution factor but rather by protein concentration and never use secondaries above 1ug/ml. Incubate for 30 min at RT.
-- Many people have complained about non-specific staining with Mol Probes secondaries though, you at least should be aware you aren't the first one to have issues. However, with the correct titration your problems should go away.
-- You may want to include BSA in your block. Use 5% for the block and 1% for the antibody diluent along with the serum. (although I have heard some people say serum actually increases background so you may want to try it without it also just to see what happens).
-- Also I find secondaries raised in donkey give much less background than goat so if you have the resources to buy new reagents you could try donkey antibodies (how about CY5?)

Also, I think Scott's suggestions of switching the A488 to your least abundant protein is an excellent suggestion. I think it makes most sense. Finally, Scott's suggestion to drop the laser power is excellent, perhaps activate a neutral density filter.

Good luck!


Hi MaximinaNYC,
will try your suggestions...