- The 6SB should comprise a sixth of the total volume in a
sample made ready for SDS-PAGE. 170μl Post-IP/5¼ 34 μl. - Leave a small volume (190–170¼ 20 μl) of the supernatant
with the IP to minimize the risk of transferring agarose beads
to the Post-IP. This small amount of supernatant is washed
away afterward. The Post-IP can be used for several Western
blots detecting the presence (not coprecipitated) or absence
(coprecipitated) of different subunit isoforms and other
proteins. - If the specific protein was precipitated from 200μg of lysate
protein and all of the specific protein was precipitated success-
fully, then adding 20μlof2SB to the agarose beads would
give a protein concentration corresponding to 10 mg/ml. This
is not the real protein concentration, but the amount of the
specific precipitated protein would be equivalent to that in
20 μl of lysate with a total protein concentration of 10 mg/ml. - The amount of lysate protein to be put into the IP and the
volume of the IP are adjustable as one finds suitable. Increasing
the amount of protein and/or decreasing the volume gives a
higher protein concentration in the Post-IP. The volume
should be sufficient to allow for proper fluid mixing in the
tube during the IP end-over-end incubation. Smaller tubes
(0.2 ml PCR tubes) are suitable for smaller IP volumes. - Since the IP only contains a “few” proteins (the precipitated
and coprecipitated proteins, plus the antibody and the protein
G from the agarose) compared to a lysate, it will often behave
differently on SDS-PAGE. Occasionally the specific protein
band shifts upward in molecular weight and does not align
with the band in the Pre-IP. Furthermore, when loading the
same amount of protein based on the input to the immuno-
precipitation, the signal intensity of the IP band is different
from the corresponding band from the Pre-IP. Often the West-
ern blot signal in the IP is lower with the exception of theγ 1
isoform, which is stronger. It is therefore advisable to load
twice the amount of protein from the IP compared to the
Pre- and Post-IPs to get similar signal intensity on the Western
blot. However, due to these and other circumstances men-
tioned above,do not quantitatively compare IP signals to the
signals of the Pre- and Post-IPs.
References
- Stapleton D, Mitchelhill KI, Gao G, Widmer J,
Michell BJ, Teh T, House CM, Fernandez CS,
Cox T, Witters LA, Kemp BE (1996) Mamma-
lian AMP-activated protein kinase subfamily. J
Biol Chem 271:611–614
2. Mahlapuu M, Johansson C, Lindgren K,
Hjalm G, Barnes BR, Krook A, Zierath JR,
Andersson L, Marklund S (2004) Expression
profiling of the gamma-subunit isoforms of
AMP-activated protein kinase suggests a major
212 Jesper B. Birk and Jørgen F. P. Wojtaszewski