1. We routinely screen 12 to 20 stable transfected MEL cell clones by SDS-PAGE in order to select a clone that expresses the tagged protein at no more than 50% of the expression level of the endogenous protein. This is to ensure that the physiological interactions and functions of the protein of interest are not disturbed as a result of the overexpression of the tagged protein.

2. The specific lysis conditions will depend on the make of blender employed. It is recommended that conditions be optimized for cell density, length of lysis time, and speed setting of the blender.

3. The final salt concentration is critical for the extraction of nuclear proteins.

4. We routinely obtain around 100 mg of nuclear extract from 4 L of MEL cell culture at a density of 2 o 106 cells/mL.

5. There are a large variety of column matrices commercially available for gel filtration, with each matrix having different optimal separation ranges and physicochem-ical properties (e.g., ability to withstand high pressure in the column). Thus, the choice of matrix will depend on the desired range of fractionation and the liquid chromatography operating system available to the user (e.g., FPLC or HPLC).

6. Users must also refer to the manufacturer's instructions and training for use of the column and the FPLC apparatus.

7. The resolution efficiency of new columns, expressed as the number of theoretical plates per meter of column under normal running conditions, should be tested first. This can be done by injecting a sample of acetone (5 mg/mL) in ddH2O water. Indicative efficiency for the analytical grade column is 11,100 theoretical plates/m.

8. While loading the extract, care must be taken that no air bubbles enter the loop. Air bubbles as well as cell debris can damage the column bed.

9. Once a new column is installed, the void (V0) volume is determined by the peak of elution of dextran blue. To further calibrate the column, a mixture of at least two proteins of known molecular weight should also be injected. Recommended standards are bovine serum albumin (67 kDa), thyroglobulin (669 kDa), and aldolase (158 kDa).

10. If there is any suspicion that the column bed has been damaged, it is best to run the calibration standards again.

11. If the blue color of the sample loading buffer turns yellow, it is because of the protein sample being acidic, which will also affect migration of the sample during SDS-PAGE. A few microliters of Tris-HCl, pH 9.0, are usually sufficient to neutralize the sample.

12. To avoid pressure buildup, the run can be started at a flow rate of 1 mL/min. It is also better to inject the sample with the lower flow rate.

13. The concentration of 150 mM KCl is critical for the efficient binding of biotiny-lated proteins to streptavidin beads. We have found that even modest increases in salt concentration severely affect binding efficiency.

14. Protease cleavage also works well with shorter incubation times (5-30 min) and a broader temperature range (4-37°C).

15. Avoid handling membrane directly; use gloves and forceps.

16. Under these transfer conditions, the temperature of the buffer can rise significantly, and frothing may occur. This does not affect the transfer.

17. The gel can be stained after blotting in order to visualize residual proteins as a test for the efficiency of transfer as well as an indication of the amount of protein loaded per lane.

18. The primary antibody can be stored and reused. Sodium azide is added to the antibody solution to a 0.02% final concentration and stored at 4°C (sodium azide stock: 10% w/v in ddH2O). Caution: sodium azide is highly toxic.

19. To reduce the risk of contaminating the samples for mass spectrometry, particularly with keratins, work is carried out in a hood with double gloves and a lab coat and always using sterile plasticware.

20. The volume of trypsin solution added will depend on the size of the gel slice. The volumes given above are for approx 4 o 2-mm gel slices. At this stage, gel slices should swell, and little solution should remain visible.


We are indebted to Dr. Jeroen Krijgsveld (Utrecht University) and Dr. Jeroen

Demmers (Erasmus Medical Center) for expert mass spectrometry analysis.

Work in our laboratory has been supported by grants from the Dutch Research

Organization (NWO), the European Union (grant HRPN-CT-2000-00078), the

NIH (grant RO1 HL 073445-01), and the Netherlands Proteomic Center.


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