4 Notes
- The IP-buffer could be the same as the homogenization buffer
or even just phosphate-buffered saline (PBS). The importance
of this is subject to a large array of optimizations. Our experi-
ence is that analyzing enzyme activity after the IP gives higher
specific activity with the milder IP-buffer compared to the
homogenization buffer described in Chapter 13 for reasons
unknown. - The Protein A or G agarose is usually supplied in a suspension
containing ethanol and has to be calibrated to the IP-buffer.
When pipetting the agarose, use wide orifice pipette tips and
vortex or stir the suspension just before pipetting. - It is paramount that the antibody used for precipitating each
subunit isoform is able to IP all of the particular subunit from
the lysate. If not all is precipitated, there is no way of knowing if
the remaining fraction is part of a special heterotrimeric com-
plex or if it is a complex fraction that is modified by PTM or
signaling that could be important for the distribution and
regulation of AMPK. Over the years, we have used many
different sources of antibodies as some have run out of stock.
Do always verify antibodies with IP and Western blot. - It is not mandatory to use^33 P-labeled ATP; the cheaper^32 P-
labeled ATP could be used instead. However, the emitted
energy from^32 P is seven times higher than from^33 P. During
the process of spotting the samples (seestep 9of the Subhead-
ing 3.2), there will be a large area with high activity radiating
your hands, and this can be minimized using^33 P instead of^32 P. - There are no special requirements for the heating block. The
96-well aluminum block can be placed on top of the inserted
blocks for tubes in the heating block, and if there is metal-metal
contact, the heat will transfer easily to the 96-well aluminum
block. - When setting up the IPs, the lysates are diluted in IP-buffer (see
step 7of the Subheading3.1). The total volume of the IPs
should be 200μl including 20μl of Protein G agarose (50:50
slurry), 10μl of antibody, and the lysate. If the lysate has a
protein concentration of 5 mg/ml, then 250μgis50μl. The
volume of IP-buffer in that IP will be
(200μl 20 μl 10 μl 50 μl¼ 120 μl). Calculate how
much IP-buffer is needed for all samples in the assay. In addi-
tion to this, all IPs must be washed in 180μl IP-buffer (seestep
16 of the Subheading3.1), and there are three IPs for each
sample, one for each heterotrimeric complex, giving 540μl
IP-buffer per sample for washing. If having 100 samples and
they on average need 120μl to fill up the first IP and 540μl for
224 Jesper B. Birk and Jørgen F. P. Wojtaszewski