Diluting/mixing of antibodies - exact method? - (Mar/18/2008 )
Hi everyone.
I am having a lot of trouble with reproducibility of a particular ELISA protocol i have developed, and now think it is possible that it may be the way i dilute the antibody (perhaps!)
What exactly is the best method of diluting an antibody? The problem i have is that it is VERY expensive, so do not like to waste it. ALso, i need to make new dilutions each time as my incubations are done in 1.5% marvel milk in PBS, so need to be fresh.
The antibody i have is supplied at 5mg/ml. This has been aliquoted into 100ul (1mg) and frozen. This stays vey stable at 4oC for a long time, so this step is no problem.
The problem is, i need to use 3ml of a 1:80,000 dilution each time i use it. So it makes sense to dilute the antibody down to 1:100 first, to create less waste. What is the best way to dilute this down? In what buffers? How should it be mixed? How should it then be stored? Is it acuurate enough to pick up 1ul to do this first dilution? if the original antibody tube should be mixed before picking any up, exactly how?
This may seem quite basic and trivial, but i really need to make sure every single step is as accurate and reproducible as possible. Everyone i talk to only seem to have vague ideas about how the antibody dilutions should be mixed, and i can't find an answer in books etc.
I really appreciate any suggestions anyone may have on this...
Thank you!
The problem is, i need to use 3ml of a 1:80,000 dilution each time i use it. So it makes sense to dilute the antibody down to 1:100 first, to create less waste. What is the best way to dilute this down? In what buffers? How should it be mixed? How should it then be stored? Is it accurate enough to pick up 1ul to do this first dilution? if the original antibody tube should be mixed before picking any up, exactly how?
This may seem quite basic and trivial, but i really need to make sure every single step is as accurate and reproducible as possible. Everyone i talk to only seem to have vague ideas about how the antibody dilutions should be mixed, and i can't find an answer in books etc.
I really appreciate any suggestions anyone may have on this...
Thank you!
first, wouldn't 100ul be 0.5mg?
your dilution should be in whatever it was first diluted with or you can use blocking buffer (if it is to be stored frozen otherwise the blocking agent (milk, in your case) can rot.
most tip and pipette combinations will not be as accurate for 1ul (if you have a combination that is accurate then disregard this paragraph). it is better and more reproducible to use larger volumes. so you may want to dilute a little more before you make your final dilution.