How to avoid signal saturation for B Actin?

[Laleh Kamalian]

Using a housekeeping gene protein like B Actin could be misleading if one is not careful about fully saturating the expression signals in Western Blot. I have tried to minimize the primary and secondry antibody concentration and incubation period. But still am not sure wheter the signals I am getting so neatly indicating same amount of protein being loaded. What is the best indicator for this? I mean how can I ensure that the B Actin signals I am getting are realy the same and not the result of fully saturation of the system?
LK

[admin]

Hello Laleh,
I know its very difficult to quantify western blots accurately. From what I know, the best way is to expose your gel to equipment which picks up the signal directly.

This method is the best for quantifying western blots however it is more time and obviously requires the right equipment, which is sometimes hard to find or expensive to buy.

If you cant get a hold of this equipment, then I suggest to try very quick exposures.
Even an instant pressing down on the cassette and taking it off quickly may get you some decent results.

How long are you exposing now in your cassette-film?

[Laleh Kamalian]

Originally Posted by admin

Hello Laleh,
I know its very difficult to quantify western blots accurately. From what I know, the best way is to expose your gel to equipment which picks up the signal directly.

This method is the best for quantifying western blots however it is more time and obviously requires the right equipment, which is sometimes hard to find or expensive to buy.

If you cant get a hold of this equipment, then I suggest to try very quick exposures.
Even an instant pressing down on the cassette and taking it off quickly may get you some decent results.

How long are you exposing now in your cassette-film?

Hi Admin
I try to increase the exposure time to 30 sec by diluting my primary antibody and reducing the incubation time. Because I'm not comfortable with too short exposur time. Poeple in my lab say the results don't seem saturated. But what is the indication?
Laleh

[kmunson779]

I think the way people in my lab do it (I don't run Westerns) is by loading different amounts of extract. Titrate the amount of protein loaded so it includes your experimental sample amount, and has at least one data point on either side. Then, quantify the loading control signal in each of the dilutions and plot it against the amount of protein loaded. If the fit line is linear over the value of the experimental samples, then you know that you haven't overloaded the Western.

Let me see if I can find a link that can explain it better. Try figure 1 of this paper, perhaps?

[admin]

Yes, I believe that method Kmunson posted is the best, but you do have to do it before you start running experiments.

If you have no choice now, try the quick exposure method, or let your membrane sit for a few hours, then try to expose it.