What dilution factor is good for Alexa 488 conjugated secondary antibody from invitrogen? I used 1:300 and getting background and autofluorescence. Is there any dilution factor that could be use for all florescent secondaries?
I use 1:1000 for AlexaFluor secondaries from Invitrogen/Life Tech.
Make sure you have secondary-only and primary-only controls and set your background threshold to those controls before imaging your samples.
I usually use 1:500, and 1:1000 for the antibody which I am using for the first time on certain cell line. As Rachel said, titration of concentration in combination with a negative control (only secondary antibody) would be the best option for new antibody.
I would say that 1:500 works for just about every fluorescent secondary if you're in a rush, but I agree with everyone else that if you have time and samples to do so, you should try several concentrations (i.e. 1:250, 1:500, 1:750, 1:1000) and see what gives you the best signal to noise ratio for your particular samples
Your secondary concentration won't alleviate the autofluorescence. I use brain tissue and had a lot of issues with autofluorescence, so now I photobleach under white light prior to starting any fluorescent staining. I also use Alexafluors at 1:500- but agree with everyone, if you're not in a rush for results try some dilution factors. Good Luck.
Usually I use 1:1000. But sometimes when my target proteins is low expressed I use 1:300 or 1:500 without background. As Rachel said, is important test some concentrations and a negative control. But is really important check if your unespecific blocking is working well... usually I use 10% goat serum or 2% BSA.
You must make serial dilutions to obtain the best concentration without any background.
it depends on the tissue and age of emryo and many factor you can begin with 1:400 or 500. if you have high background you have to use a lowre concentartion
When you do positive or negative control you incubate samples with PBS or complete media!
Doaa Yamani You should incubate all of your samples in the same solution +/- the things you are controlling for (e.g., primary antibody).
@ Stephanie M. Miller
Could you explain more how do you photo bleach under white light!
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