Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 174

1. Rotation wheel
2. Bench top centrifuge

5.1.2. Instruments

5.2. Procedures

1. In order to maintain the protein-protein interaction integrity,
the total protein lysate and the immunoprecipitation proce-
dure is carried out at 4°C.
2. To initiate immunoprecipitation, we use at least 200 mg of total
protein from the total protein lysate and dilute tenfold with
TBST.
3. Then, 3-5 mg of the specifi c antibody is added to the diluted
protein lysate ensuring that the antibody is in excess of the
target protein of interest. As a negative control, no antibody is
added to the same amount of diluted protein lysate.
4. This sample mixture is then transferred into a 1.5-ml centri-
fuge tube and all the samples are placed into a rotation wheel
prechilled at 4°C in the cold room.
5. After incubating for at least 1 h, a slurry containing 20 ml of
protein-A cross-linked to sepharose or agarose beads are added
to the sample mixture.
6. The fi nal sample mixture is placed onto the rotation wheel
again for another 1 h.
7. The sample mixture is then subjected to centrifugation
(14,000 × g , 4°C, 1 min) with the supernatant decanted
leaving the sepharose or agarose beads.
8. The sepharose or agarose beads are resuspended in 0.5 ml of
TBST before undergoing centrifugation (14,000 × g , 4°C,
1 min).
9. Step 8 is repeated between three and fi ve times.
10. Finally, the beads are resuspended in 50 ml of Sample Buffer
and heated to 95°C for 3 min.
11. The samples are then ready to undergo Western blot analysis as
described in Sect. 3 .

5.3. Variations

In cases when a suitable antibody for immunoprecipitation is not
available, the common practice is to add a known tag such as myc
or fl ag onto the N- or C terminal of the gene encoding the protein
of interest. Then the fusion gene is transfected into a cell line before
the immunoprecipitation begins. In this way, the proteins associat-
ing with the protein of interest can also be identifi ed. Nevertheless,
the caveat of this approach is the potential inclusion of artifact
proteins that bound to the over-expressed fusion protein with no-
physiological signifi cance. Also, recently, a new type of magnetic
beads is commercially available to replace the sepharose or agarose
beads. The advantages of the magnetic beads is in their shorter