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antibody in 40% glycerol


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6 replies to this topic

#1 jasmina

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Posted 12 October 2011 - 03:05 AM

I want to do western blot of protein for which the antibody should be diluted in 40% glycerol. Is it correct to use the following method of calculation?
I divide final volume of incubation per dilution factor and then, multiply per 1.6.

Thanks for your help

#2 Papaver

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Posted 12 October 2011 - 06:00 AM

Why multiply per 1.6?

#3 jasmina

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Posted 12 October 2011 - 07:07 AM

becos in a case of antibody in 50% glycerol, i multiply by 2.

#4 almost a doctor

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Posted 12 October 2011 - 07:33 AM

I think there's a bit of confusion here, I too don't really understand what you refer to with the 1.6.
I assume the multiplying by 2 is so that is a 1:2 dilution (or 50%), which will be fine assuming the 50% is v/v.

What I don't really understand here is what are you multiplying by 2 (or 1.6), and what exactly you refer to with the dilution factor and final dilution, is the Ab, the glycerol, both.... I mean, if you prepare your Ab (which is what you will consider final volume and dilution factor for) and then multiply by 1.6 (or 2), isn't that going to change your antibody concentration?

Can't you just prepare a 40% glycerol solution, and use that to dilute your Antibody? I am assuming here the amount of Antibody you need to add is low enough that it is not going to affect your glycerol concentration. So say, you need a 1:2000 Ab dilution. Just add 1ul to 2ml of 40% glycerol.

Or am I missing something???

#5 jasmina

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Posted 12 October 2011 - 07:52 AM

I have already aliqots of antibody dissolved in 40% glycerol! so, I have mix of antibody with glycerol.
If I need to use for example 1/2000 dilution in 6ml of blocking solution, so what I do is taking 3ul from the whole aliqot (mix) and multiply by 1.6, so I take 4.8ul from the mix and add to the 6ml of 5% milk in PBStween
is this correct??
thanks

#6 almost a doctor

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Posted 12 October 2011 - 08:39 AM

Hi, yes that calculation will be okay. However, unless you know the actual concentration of Ab you want, and the concentration in the 40% glycerol stock (ie, mg/ml of Ab), I'm not sure is really necessary to do it that way. I mean, if you are using a 1:2000 (to follow the example) of your stock, and that works, do you really need to calculate a 1:2000 form the Ab itself?
Maybe you do for your particular WB, but I in my opinion unless you are working with actual mg/ml (or absolute amount of Ab), I don't think you need to be that precise in this case.

Now, if this is to get the same amount of Ab you were using before with the 50% glycerol, maybe you really need to be that accurate.

#7 jasmina

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Posted 12 October 2011 - 12:24 PM

I get your point! but, in my case, the concentration of antibody is not precised in the datasheet! its whole serum, should be dissolved in 40% glycerol!
so, to be sure about antibody amount I did for my incubation, I did the previous calculation!
thanks for your interaction




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