Different Dilution factors for the normalizator

[sotellme]

I am looking for some ideas about this one. I use B actin as the normalizator in my western blot. i use different dilution factors of the B actin antibody. For some samples i use 500X and for others i use 1000X. I wonder when i normalize my samples and calculate the relative expression levels for the different protein targets should i take the different dilution factors of the B actin in account or should i pretend that they have the same dilution factor?

Thanks.

 

[Moonbear]

You shouldn't use different dilutions at all. There is no conversion factor you can use, because antibody binding is not linear, so if you've used different dilutions of antibody, that's going to be useless to normalize your samples. Why are you varying your antibody dilutions in the middle of an experiment? If you're changing your technique, nothing is standardized.

Before you run any more experimental samples, sit down with someone who KNOWS this technique and plan out what you need to do and work with them, otherwise you're going to just keep wasting samples and money and will have completely useless data. If nobody in your lab knows how to do this, plenty of people do Westerns all day, every day; visit someone else's lab to learn. It's a lot cheaper to pay the travel expenses to visit another lab than it is to keep throwing away samples by making mistakes anyone who knows the technique could have stopped you from doing in the first place.

 

[sotellme]

Dear Moonbear!

You saved my life! actually this is the idea of my advisor. He told me to use different dilution factors. I need to have a talk with him. Thank you!