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pondering on antibody dilution

pondering on antibody dilution - Western Blot Forum

pondering on antibody dilution - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 03-18-2011, 01:59 AM
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Lightbulb pondering on antibody dilution



will the dilution of the antibody will be decided based on the protein loaded in the gel or based on the suface area of the blot..?
i am confused because sometimes i may load protein in just one lane and sometimes in all lanes. In that case will antibody dilution remains the same?
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Old 03-18-2011, 06:59 AM
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Default Re: pondering on antibody dilution

Hola, following the data sheet of the ab. you will have an excess of ab, only you have vary the volume of solution in function of the size of blot. If ab are home made you have titering it but 175oo for rabbit serum and 1/2000 for mouse will be enough for some experiments. Buena suerte
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Old 03-21-2011, 11:58 AM
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Default Re: pondering on antibody dilution

I have a similar question. Last time I was doing western blot and I loaded 15 ul of my samples on gel, I got some pretty good results.. my primary Ab was diluted,1:2000, and my secondary 1:5000. Then I went with 10 ul of samples on the gel and I repeated the whole protocol in the same way like the last time, and got nothing. Can it be caused by the fact I loaded only 5 ul less of the sample then the last time<?
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