Protein Protocols Protein Protocols

Immunoprecipitation 1105
2. Dry the gel onto a 3-mm Whatman paper, presoaked in water, in a gel dryer at 60°C for
2–6 h (see Note 11).
3. Place the gel together with an X-ray film in a light-excluding X-ray film casette at
–70°C and expose the film for an appropriate time (days–weeks) (see Note 12).
4. Develop the film in an automatic X-ray film processor or manually for 5 min each as
follows (see Note 13): developer, water bath, fixative, and running water.
4. Notes
4.1. Metabolic Labeling of Proteins
1. When radiolabeling cells for longer than 6 h, all the radiolabeled amino acids may be
consumed. It is therefore sometimes necessary to add unlabeled amino acids, i.e.,
methionine and/or cysteine as shortage of amino acids may cause an interruption in protein
synthesis.
2. If small volumes are used during labeling, keep the flask on a slow rocker or shake the
dishes every 15 min to ensure that the cells do not dry.
4.2. Lysis of Cells
3. To optimize the extracting conditions, try a stronger lysis buffer on the centrifuged cell debris
than the buffer you initially used for lysis of the cells. Then test by immunoblotting whether or
not most of the labeled proteins were extracted with the lysis buffer initially used.
4. To remove aggregates of cytoskeleton elements after thawing of lysate, centrifuge samples
at 12.000g for 5 min.
5. Caution: PMSF is extremely destructive to mucous membranes of the respiratory tract,
the eyes and skin. Also, the other protease inhibitors are toxic.
4.3. Formation of Immune Complexes
6. To avoid nonspecific binding of complexes to the tube wall, use good quality tubes (e.g.,
Eppendorf, Hamburg, Germany) or pre-coat the tubes with 0.5% bovine serum albumin
for 15 min.
7. Before heating samples to 100°C, use a needle to make a small hole in the tube cap. This
will prevent the building of excess pressure in the tube and the tube cap will remain closed
during the heating step.
4.4. SDS-PAGE
8. Caution: Acrylamide and bis acrylamide are neurotoxic and may be absorbed through the
skin. Polyacrylamide is considered nontoxic but may contain unpolymerized material.
9. When preparing and handling gels, use gloves to avoid exposure to unpolymerized polyacrylamide
and radioisotopes.
4.5. Detection and Analysis of Immunoprecipitated Proteins
10. Caution: Salicylate may elicit allergic reactions and is readily absorbed through the skin. As
an alternative use commercial fluorescents available from several companies.
11. Shrinkage, distortion, and cracking of the gel are common problems encountered when
trying to dry gels. To avoid shrinkage and distorsion, dry the gel onto a 3-mm Whatman
paper (presoaked in water). Make sure there are no air bubbles between the gel and the
paper before starting the gel dryer. To avoid cracking do not turn off the gel dryer or break
the vacuum before the gel is completely dry. If possible, use thin gels (0.75 mm), since
cracking is more common with thicker gels containing larger amounts of polyacrylamide.