washing, then this is a total of (120μlþ 540 μl) * 100¼66 ml
of IP-buffer. Approximately 10 ml of IP-buffer will be used for
the initial washing and calibration of the Protein G agarose, and
then always make a little extra. Thus, for this assay with
100 samples, make up 90 ml of IP-buffer.
- The selection of either Protein A or G agarose is dependent on
the species and subgroup of the antibody used in the IP. Most
antibodies work best with Protein G, but this should be verified
before starting. As standard we use Protein G agarose, Fast
Flow from Merck (cat. #16-266).
- The amount of antibody to be used in an IP depends on the
amount of lysate put into the IP and the affinity of the anti-
body, which must be tested. Usually 2μg of antibody is suffi-
cient for IP on 200μg lysate. The concentration of antibody
varies between batches, and to avoid pipetting very small
volumes, it is recommended to dilute the antibody in
IP-buffer to a concentration of 0.2 mg/ml and then pipette
10 μl to each IP.
When doing consecutive IPs on AMPK, it is very important
to do the IPs in the right sequence. Working with human
muscle samples, only three different heterotrimeric complexes
have been identified [10, 11]. Theβ2 subunit is part of all three
complexes, so the IPs cannot be done with aβ2-specific anti-
body. Theα2 subunit is part of two of these complexes, so
starting with anα2 IP would pull down both these complexes
at once. The trick is to IP theγ3 subunit first isolating the
α 2 β 2 γ3 complex. Thenα2 can be precipitated isolating the
α 2 β 2 γ1 complex. The only complex left in the supernatant
then is theα 1 β 2 γ1 complex, which can be isolated with an
α1IP.
Working with mouse muscle makes things a little more
complicated, because there are five complexes [12]. All five
complexes cannot be isolated with consecutive IPs on the
same lysate sample. Two sets of consecutive IPs must be set
up as follows to get the individual activity of all five complexes:
Set1: IP1γ3(α 2 β 2 γ3), IP2β1(α 1 β 1 γ1 andα 2 β 1 γ1), IP3
α2(α 2 β 2 γ1), and IP4α1(α 1 β 2 γ1).
Set 2: IP1γ3(α 2 β 2 γ3), IP2β2(α 1 β 2 γ1 andα 2 β 2 γ1), IP3
α2(α 2 β 1 γ1), and IP4α1(α 1 β 1 γ1).
The proportion ofβ1 complexes is however quite small and
likewise the activity, so it might not be worth the effort to try to
isolate theβ1 complexes from theβ2 complexes, unless specifi-
cally interested in theβ1 activities. Just doing three consecutive
IPs like on human lysates (γ3,α2,α1) would make the assay
much simpler, and one might acquire the sufficient level of
information.
Kinase Activity of Specific Heterotrimers 225
