fluorescent secondary antibodies --> reccomendations? - (Dec/11/2007 )
Hi everyone,
I am new to this forum. I have been working with immunofluorescence for some time and have a primary raised in mouse that I use. I originally was using alexa594 goat-anti-mouse from molecular probes and never had any problems with background/non-specific binding.
I am now trying colocalization with another primary raised in goat, so I bought -- alexa594 rabbit anti mouse and alexa488 donkey anti goat, so I don't get cross reactivity. (both from molecular probes)
I am getting tons of background and very bad non specific binding, I have tried a number of different blocking methods (BSA,FBS, even skim milk :S ), as well as tons of different dilutions, I always spin down my secondaries etc. My original secondary always works but these ones dont.
I've been in contact with tech, help there and so far they have been pretty useless, looks like these antibodies just aren't any good.
I was wondering if anybody has success with other secondaries from this company or other companies that they have found reliable. I need fluorescent conjugated secondaries that binds goat IgG and one that binds mouse IgG, that won't cross react and that are different colours.
Thanks so Much!
I assume that when you were trying to figure out what is the problem with the new Abs, you used the old one as control and found that only the new ones got background.
Not very sure what happened with these Abs, but I always use Jackson's secondary antibody. Nowadays I use the donkey set, all donkey Ab against other species like mouse, rabbit, goat... I use FITC, Cy3 and Cy5 conjugated and am happy with them. You may want to give it a try.
Not very sure what happened with these Abs, but I always use Jackson's secondary antibody. Nowadays I use the donkey set, all donkey Ab against other species like mouse, rabbit, goat... I use FITC, Cy3 and Cy5 conjugated and am happy with them. You may want to give it a try.
Yes I always use the old antibodies as a control, with and without primary to make sure I am getting specific binding and no background, it is a very frusterating problem since the old ones work.
What filter sets do you use for Cy3 and Cy5? and what kind of dilutions do you have to use?
your secondaries sound fine - have you tried blocking with the serum of the animal it was raised in (also do the staining in series not in parralel)
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yes I've tried blocking with the animal serum, and I've done both series and parallel. The company agrees based on the pictures I sent them that the secondaries are no good and are crediting me. They also said that those particular antibodies do have background and non specific binding problems (annoying). I just want to know which antibodies people have found reliable.
the very ones you mentioned
theres always the avidin biotin route i suppose but that might create more problems than it solves
I dilute them 1:100 or 1:200, but my understanding is that they can be used at lower concentration, 1:500 or so but I never try. Both are bright, stable, low background. Cy3 is maximally excited at 550nm, peak emission at 570; Cy5 is 650 and 670, respectively.
Here are from Jackson bookset: For Cy3: Can use whatever filter set similar to TRITC set (the rhodamine dye), using either argon laser (514nm or 528nm lines), 50% of maximum; or 75% of maximum with a helium/neon laser (543nm) or mercury lamp (546nm). If use together with FITC, should set narrow band-pass emission filter for FITC to minimize bleed through of Cy3. I have never got any problem with this. I can see (by eyes) the red channel just by using the green channel (kind of orange), but it is no problem when taking picture.
For Cy5: use about 98% of maximum with krypton/argon laser (647nm) or 63% of helium/neon laser (633nm).