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Regulation and Therapeutic Potential of the PI 3-kinase/PKB Signalling Pathway.

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Immunoprecipitation of Proteins


[Note: If using a pre-existing cell lysate, proceed directly to step 5.]

1. Wash adherent cells twice in the dish or flask with ice-cold PBS and drain off PBS. Wash non-adherent cells in PBS and centrifuge at 800 to 1000 rpm in a table-top centrifuge for 5 minutes to pellet the cells.

2. Add ice-cold modified RIPA buffer to cells (1 ml per 107 cells/100 mm dish/150 cm2 flask; 0.5 ml per 5 x 106 cells/60 mm dish/75 cm2 flask).

3. Scrape adherent cells off the dish or flask with either a rubber policeman or a plastic cell scraper that has been cooled in ice-cold distilled water. Transfer the cell suspension into a centrifuge tube. Gently mix the suspension on either a rocker or an orbital shaker at 4íC for 15 minutes to lyse cells.

4. Centrifuge the lysate at 14,000 x g in a precooled centrifuge for 15 minutes. Immediately transfer the supernatant fraction to a fresh centrifuge tube and discard the pellet.

5. To prepare protein A or G agarose/sepharose, wash the beads twice with PBS and restore to a 50% slurry with PBS. It is recommended to cut the tip off of the pipette tip when manipulating agarose beads to avoid disruption of the beads.

6. Pre-clear the cell lysate by adding 100 microliters of either protein A or G agarose/sepharose bead slurry (50%) per 1 ml of cell lysate and incubating at 4íC for 10 minutes on a rocker or orbital shaker. Pre-clearing the lysate will reduce non-specific binding of proteins to the agarose or sepharose when it is used later on in the assay.

7. Remove the protein A or G beads by centrifugation at 14,000 x g at 4íC for 10 minutes. Transfer the supernatant to a fresh centrifuge tube.

8. Determine the protein concentration of the cell lysate, e.g. by performing a Bradford assay. Dilute the cell lysate at least 1:10 before determining the protein concentration because of the interference of the detergents in the lysis buffer with the Coomassie-based reagent.

9. Dilute the cell lysate to approximately 1mg/ml total cell protein with PBS to reduce the concentration of the detergents in the buffer. A more concentrated cell lysate (i.e., 10 mg/ml) may be necessary to immunoprecipitate a protein which is found in low levels in a cell model.

10. Add the recommended volume of the immunoprecipitating antibody (see Certificate of Analysis for detailed information) to 500 microliters (i.e., 500 micrograms) of cell lysate. The optimal amount of antibody that will quantitatively immunoprecipitate the protein of interest should be empirically determined for each cell model.

11. Gently mix the cell lysate/antibody mixture for either 2 hours or overnight at 4íC on a rocker or an orbital shaker. A 2 hour incubation time is recommended for the immunoprecipitation of active enzymes for kinase or phosphatase assays.

12. Capture the immunocomplex by adding 100 microliters protein A or G agarose/sepharose bead slurry (50 microliters packed beads) and gently rocking on either a rocker or orbital shaker for either 1 hour or overnight at 4íC. In many instances, immunocomplex capture can be enhanced by adding 2 micrograms of a bridging antibody (e.g., rabbit-anti-mouse IgG). This is especially important with antibodies that bind poorly to protein A such as mouse IgG1 or antibodies generated in chicken.

13. Collect the agarose/sepharose beads by pulse centrifugation (5 seconds in the microcentrifuge at 14,000 rpm). Discard the supernatant fraction and wash the beads 3 times with 800 microliters ice-cold modified RIPA buffer. Occasionally, washing with modified RIPA buffer will strip the immunocomplex from the agarose/sepharose beads. In these cases, washing with the milder PBS is recommended.

14. Resuspend the agarose/sepharose beads in 60 microliters 2 x sample buffer and mix gently. This will allow for sufficient volume to run three lanes.

15. The agarose/sepharose beads are boiled for 5 minutes to dissociate the immunocomplexes from the beads. The beads are collected by centrifugation and SDS-PAGE is performed with the supernatant fraction. Alternatively, the supernatant fraction can be transferred to a fresh microcentrifuge tube and stored frozen at -20íC for later use.  Frozen supernatant fractions should be reboiled for 5 minutes directly prior to loading on a gel.



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Preparation of Modifed Radioimmunoprecipitation (RIPA) Buffer

Activation of Sodium Orthovanadate

Preparation of Cell Lysate

Western Immunoblotting of Proteins

Troubleshooting Tips for Western Immunoblotting

Immunoprecipitation of Proteins

Immunoprecipitation / Kinase Assay

Troubleshooting Tips for Immunoprecipitation / Kinase Assays

Assay for Immunoprecipitated Phosphoinositide 3-Kinase (PI 3 Kinase) Activity (in vitro)

Phospholipid Vesicle Preparation

Chromatin Immunoprecipitation (ChIPs) Assay Kit

Forskolin Treatment of Cells

Preparation of Brain Cytosol

Immunohistochemistry with Fixed Paraffin-Embedded Tissue Sections

Tips for Immunohistochemistry with Fixed Paraffin-Embedded Tissues

Immunohistochemical Methods to Detect Nitrotyrosine

Immunocytochemistry with Adherent Cells

Differentiation of Stages in the Cell Cycle

Elution of GST-Fusion Protein from Glutathione Agarose

Purification of Antiserum or Ascites by Protein A/G Chromatography

Molecular Weights of Some Common Proteins

Transient Transfection of Cos-1 Cells

The Genetic Code
 
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