I have never done IP before and need some advice on the experiment planning:
I will collect cell lysate and incubate it with an antibody of my interest. The antibody is rabbit polyclonal IgG. I will use the Protein G magnetic beads to do the IP, and do western blot to detect the amount of this particular protein in wt and mutant samples. My naive questions are:
1. Can I use the same antibody for IP and western?
2. I would like to include beta-actin control in the western. Can I do IP with beta-actin and the other antibody at the same time or I have to do them separately?
3. What kind of control should I include? I am thinking about total cell lysate; total cell lysate with IP but no antibody; what else is needed?
Thank you.
1. In many cases you can use the same antibody for western as you did for IP, but not all. Since the IP will be done when the target protein is in its native conformation, and the western will (I'm assuming) be done under denaturing conditions, the epitope recognized by the antibody has to be available in both cases in order for it to work. It would be best to check whether the manufacturer of the antibody has tested it for both applications.
2. Housekeeping proteins like beta actin or tubulin are typically not immunoprecipitated for use as a loading control (though of course they could be used as a loading control for the whole-cell lysate or pre-cleared lysate). Rather, loading is normalized by the abundance of the protein of interest in your IP.
3. In addition to your whole-cell lysate, it would be good to include an antibody isotype control (an IP done in parallel using an antibody with irrelevant specificity) - so in your case you could use purified pre-immune rabbit polyclonal IgG (can usually be bought companies that sell antibodies.
1. In many cases you can use the same antibody for western as you did for IP, but not all. Since the IP will be done when the target protein is in its native conformation, and the western will (I'm assuming) be done under denaturing conditions, the epitope recognized by the antibody has to be available in both cases in order for it to work. It would be best to check whether the manufacturer of the antibody has tested it for both applications.
2. Housekeeping proteins like beta actin or tubulin are typically not immunoprecipitated for use as a loading control (though of course they could be used as a loading control for the whole-cell lysate or pre-cleared lysate). Rather, loading is normalized by the abundance of the protein of interest in your IP.
3. In addition to your whole-cell lysate, it would be good to include an antibody isotype control (an IP done in parallel using an antibody with irrelevant specificity) - so in your case you could use purified pre-immune rabbit polyclonal IgG (can usually be bought companies that sell antibodies.
Hi there,
What you may also do to avoid non specific interaction is to perform a pre clear on the lysate by incubating it with beads in order to eliminate proteins in the lysate interacting non specifically with the beads.
Hello
I agree with the points of Max and Dominique. You may also add some non-specific serum or antibody to the beads during the pre-clearing.
In addition, I hope your target protein does not migrate in the surroundings of 25 or 50 kDa, because this is where the light chain and the heavy chains of your immunoprecipitating antibody will show up.
If your target protein should co-migrate with LC or HC, it would be an advantage to have two antibodies made in different species (rabbit and mouse, most commonly). Then the species-specific secondary antibody in the Western will only detect the relevant primary (Western) antibody, not the IP antibody . If this is not possible, you may have to covalently link the IP antibody to the beads.
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