# Antibody question - (Dec/05/2006 )

We're having a bit of a discussion of this in our lab and does the actual volume (not concentration) of antibody have any affect on the outcome of a western? And I apologize if this seems basic.

For instance if an antibody has an extremely high binding affinity and 1:50,000 dilution is sufficient for a western. Would 1 ul into 50 mL of blocking solution have the same affect as 1 ul of antibody diluted into 1 ml (1:1000) and then 250 ul of the 1:1000 dilution into 5 mL for a final of 1:50,000 have the same western blot result?

I would think that the final concentration they would be the same and the actual volume shouldn't have an affect. Would that be correct?

-cancergeek-

QUOTE (cancergeek @ Dec 5 2006, 10:05 PM)
We're having a bit of a discussion of this in our lab and does the actual volume (not concentration) of antibody have any affect on the outcome of a western? And I apologize if this seems basic.

For instance if an antibody has an extremely high binding affinity and 1:50,000 dilution is sufficient for a western. Would 1 ul into 50 mL of blocking solution have the same affect as 1 ul of antibody diluted into 1 ml (1:1000) and then 250 ul of the 1:1000 dilution into 5 mL for a final of 1:50,000 have the same western blot result?

I would think that the final concentration they would be the same and the actual volume shouldn't have an affect. Would that be correct?

har har, you will test us, wonÂ´t you, or being weak in mathematics?

the first dilution is actually 1: 50.000,

the second, however, is 1:1000 x 1:20 = 1:20.000,

so both are not equal
q.e.d.

-The Bearer-

haha my bad. I meant 100 ul of the 1:1000 into 5 mL (1:50 x 1:1000). Would that give comparable results as a straight up 1:50,000 or 1 ul into 50 mL.

-cancergeek-

why would it not have the same effect? there is the same amount of antibody in 10 mL of your solution at the end? Unless the antibody remember how it was diluted?
the memory of the antibody has not yet been proved

-Missele-

I think the question was if a 1:50,000 dilution in 50 ml would give the same results as a 1:50,000 in 5 ml as final volume for the blot to be incubated in ... if I am wrong, than it's wishful thinking because I'd like to know the answer to that question too

-hann.a-

it would be the same the first time you used it. after that it will not be the same if reused. the 5 ml will deplete faster than the 50 ml.

-mdfenko-