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antibody-dilution???

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Kerstin:
Hi everybody,

I´m working with immunocytochemistry for some month now and I´m wondering how the antibodies are really diluted: In nearly every lab there is another method!
When the data-sheet is saying the working dilution for the antibody is 1:1000, what are you doing?
Do you just take one part of the given antibody and 999 parts of your dilutent? Or do you calculate with the given protein concentration of the antibody (eg 2mg/ml)...?

thanxx for your help   Kerstin

immunoem:
Hi Kerstin,
antibodies tend to fall into two groups - those where the Ab concentration is known (typically mAbs and purified pAbs) and those where the Ab concentration is now known (as with immune serum). Where the stock Ab concentration is known, the working concentrtion should really be stated (typically in range of 0.1-10 ug/ml for most Abs). It's a bit naughty to just say 1:1000 dilution where the stock concentration is known as not all batches of Ab are exactly the same concentration. Conversely, if the Ab concentration has not been determined then you have to go with a dilution factor such as 1 in 1000, etc.
I hope this makes sense.
Ian

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