Normalizing Western Blot to cell number without cell counting - (Apr/04/2006 )

Hi!

I got the following problem: I want to have a look at protein expression in primary culture of neurons during their development.
I would like to know if someone has any idea, which protein I can use to have a kind of loading control which is not related to total protein amount but to the cell number. Which means a protein that is not upregulated during the growth of the cell. I thought about histones? Do you have better suggestions? maybe a protein with higher MW than the histones? Unfortunately i cant count my cells due to their culturing conditions.

Thanks a lot!
Mike

You may find a literature search helps you here.

What about the classics? Beta-actin, GAPDH, TBP, etc. Not being a neuronal development expert I do not know how the expression of the genes is affected by growth. I guess beta-actin expresion will increase as the cell gets larger as it has to support larger networks. Shame, it's a synch to blot.

Actin and Tubulin increases unfortunately. I also thought about GAPDH and TBP. But I guess they will increase, because they are more or less increasing with the total amount of protein, as there will be more metabolism or transscription in a bigger cell. Unfortunately, I didn't find something in the literature. But I will also try GAPDH. But I guess it will be more a loading control for the amount of protein loaded and not related to the cell number.
Thanks for your help!

does the nucleus continue to grow along with the rest of the cell? if not, could you use a structural protein of some sort specific to the nuclear membrane?

the nucleus seems also to grow, if one looks to the microscope images.
I go now for a histone 3 antibody. The chromatin ammount should stay the same in a postmitotic cell.
Thanks for your replies!

You can try Lamins,
Lamins are proteins in the nuclear envelope.
I am not sure if there are more lamins in bigger cell, but you can try to find out.
Some antibody manufactures sell antibodies to lamins as an official nuclear-protein-level loading control.
Gr. Eveline