Antibody Dilution - (Feb/08/2007 )

How do you know how much to dilute the primary antibody? I usually go for 1:1000

Thanks

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trial and error.

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The antibody dilution depends on the type of assay you are performing. For what assay are you diluting your antibodies for?

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I am doing for westerns. Do you go by the manifacturers reccomendations?
Or you look at papers which have used it.

And how do you do trial and error. I am assuming you load the gels and cut it in halves and try different dilutions on each half.

thanks

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First you try the manufacture recommedation
...if high background... increase dilution
....if the bands are too weak...decrease dilution.



yes, if you want to find the optimal dilution of antibody for the western

Hope this help.

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Have the manufacturer's recommendations as a starting point. Try to look at papers which might have used them.

And depending on what results you get, you can decide of you want to increase or decrease the concentration.

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Today I tried different dilutions of the antibody( 200,500, 1000) and I got a band for all diltuions for the positive control. But none for the samples. So does it mean that I should load more protein?

I was looking for caspase-3 ( pro caspsae and caspase).

Thanks

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If you could see a clear band with a 1:1000 dilution, try 1:2000 or 1:5000. Its good for you as you are saving more antibody.

but as you say that the samples dont have a band, try a 1:100, but then think of the possibility that there is no caspase-3 or that its too low for detection. Try loading more sample and check it.

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[attachment=2564:caspace3_200.jpg]These are my band for !;200 (1),



1:500.[attachment=2566:caspce3_500.jpg]

The darkest lane ( end lane) is a positive control for capase-3 ( 35 kda). I am thinking of using 1:200 since I can see cleaved caspase in my samples.

I am not sure what to read abt the unspecific bands. The antibody says it cross reacts with other caspases. Any opinions are welcome

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Just make sure the bands you are seeing with 1:200 are cleavage bands and not unspecific bands.

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