Biomedical Engineering Reference
In-Depth Information
4
Notes
1. We have found that antibodies directed against epitope tags
such as FLAG perform far better in immunoprecipitations than
those directed against specifi c proteins.
2. ATP must be added fresh each time. BSA need only be included
in the kinase assay with Akt, and should also be added fresh.
We have found that PI can interfere with the reaction and
should be used very sparingly.
3. Other recombinant active kinases could similarly be used.
4. It is important to use TBST rather than PBST when probing
for phosphorylated proteins, as the phosphates can interfere
with detection. To improve sensitivity, antibodies should always
be incubated overnight. We also use six 5 min washes for phos-
phorylated antibodies as this improves specifi city.
5. BSA should be used for blocking as there are phosphorylated
proteins in skim milk which can interfere with detection.
6. Phosphorylated Akt substrates antibody and other phosphory-
lated substrate antibodies are available from Cell Signaling
Technology. Note that the specifi cities of these antibodies can
be exploited to your own advantage—for example, we have
used the fact that phosphorylated Akt substrates antibody
detects the phosphorylated form of S6 [
18
] as a control [
19
],
and strengthened our fi ndings by using two versions of the
phosphorylated Akt substrates antibody [
20
].
7. Other detection methods could also be used.
8. Any cell type that provides high transfection effi ciency and
protein may be used.
9. At this point, a protein assay may be used to ensure equal
amounts of protein are used in multiple samples.
10. Thorough washing of the protein G-Sepharose beads in RIPA
is necessary beforehand as the commercial stock is preserved
in ethanol. To facilitate pipetting of the sepharose, we recom-
mend cutting the ends off tips.
11. The amount of antibody used here can be titrated.
12. The supernatant can be subjected to Western blotting to assess
immunoprecipitation effi ciency.
13. These ratios are required to ensure that the proteins refold cor-
rectly when moving from one buffer to another.
14. Very small magnetic stirrers are required here to ensure mixing
in the 2 ml tube.
15. We use a mixture of two parts RIPA buffer, two parts 10 %
SDS, and one part 5× loading buffer.
