Simple dilution question - (Oct/25/2007 )

Hi everyone,
I'm very sorry for being so stupid, but I just want to make sure this is dilution is correct before i set up my ELISA. I always get really confused when working with such small amounts.

My antibody is supplied at 10 mg/ml or therefore 10ug/ul

I would like to coat each well of my ELISA plate with 50 ul containing a total of 50 ng. Or, 1 ng/ul needing about 4 ml in total.

If i take 1 ul of my original antibody, and dilute in 1 ml, this gives a conc of 10ug/ml or, 10ng/ul.

so if i take 1ul of this in 100ul, this will give 1ng/ul ???

so if i therefore need 4 ml, i take 40 ul into 4 ml?

This doesnt seem right....

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do a 10 times dilution of the antibody (10 uL plus 90 uL of buffer) : 1 mg/mL
you want 4 mL at 1 ug/mL. So you have to dilute 1000x your solution at 1 mg/mL. It means you have to take 4 uL of antibody (1mg/mL) into 4 mL.

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My antibody is supplied at 10 mg/ml or therefore 10ug/ul

I would like to coat each well of my ELISA plate with 50 ul containing a total of 50 ng. Or, 1 ng/ul needing about 4 ml in total.


You have 10ug/uL, and you want 1ng/uL, or .001ug/uL.

So, divide: 10ug*uL^-1/.001ug*uL^-1 to find the dilution factor.

Units cancel, giving dilution factor of 1:10,000

Therefore, 1uL antibody in 10mL water gives proper dilution and concentration. Then simply use 4 mL of that solution. ( I would not try to pipette less than 1uL, as it rarely is very accurate)

Best of luck!

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Gosh i am stupid when it comes to these...

I think i understand gunnerpiaffe's but not missele's...

taking 10 ul of original into just 90 ul, wouldnt that give 1mg/100ul?

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taking 10 ul of original into just 90 ul, wouldnt that give 1mg/100ul?
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This is a 1:10 dilution of the original. So 10ug/uL becomes 1ug/uL.

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it's a 10 time dilution. so 10 mg/mL diluted 10 times gives 1 mg/mL. Then you still have to dilute 1000x.


Actually you need to dilute 10.000 times the antibody to get the working dilution. So the solution of gunnerpiaffe is the easiest one.

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Brilliant,
thanks for your help on this. I don't know why I cant get my head round it!

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".....giving dilution factor of 1:10,000

Therefore, 1uL antibody in 10mL water gives proper dilution and concentration....."

hello gunnerpiaffe, i`m a bit confused here with the calculation.
if the dilution factor is 1:10,000, i think it means 1 ul of antibody in 9,999 ul (or 9.999 mL) water to get a total volume of 10,000 ul
or, is it possible that what you meant by 10 ml is 9,999 ul?

forgive me if I`m wrong

audrey

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Meticulously speaking, you are right. Dilution of 10000 times means that 1ul of Ab in 9999ul of buffer (for example). But in this situation, there is not necessary for THAT accurate. So 1ul in 10000ul (or 10ml) is fine and that is what most people use.

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