gel with 3 M yellow electrical tape. Tape must be removed before running the gel. Four second dimension gels can be poured by using an extra set of spacers and a sandwich divider plate between the outer plates when assembled in the casting stand.
Frozen protein samples should be thawed at room temperature and vortexed before loading. If a precipitant exists in the protein sample then it can be heated at 40°C for 5 min. The samples should not be boiled or reducing agent added to them.
A cutting tool can be made by gluing or taping four No. 9 single edged industrial razor blades on both sides of a 1.5mm thick spacer (see Fig. 12.1).
Place a few drops of 1X running buffer on top of the gel slice to help it slide down the side of the glass plate. It may be necessary to use the long side of 1 mm spacer to uniformly push the slice into the bottom of the well and prevent the gel slice from flipping on its side.
For all steps involving manipulation of gels, it is essential that gloves are worn and frequently changed and that the staining boxes are thoroughly cleaned with rinsable detergent and washed extensively with water to avoid contamination of gels with skin keratin as this will complicate mass spectrometry analysis.
The Redox 2D-PAGE technique preferentially results in the detection of abundant proteins in the 40-200 kDa range. Less abundant proteins in the 15-40 kDa range can be detected by developing the silver stained gels for longer periods of time at the expense of a higher background in the upper portion of the gel. Increased resolution of a specific molecular weight range of proteins can be achieved by varying the percentage of acrylamide accordingly in the first and second dimension gels. The number of DSBP is lower in the nucleus compared to the cytoplasm so more protein can be resolved in the first dimension and longer developing times may be required following silver staining for detection of spots (see Fig. 12.2)
To obtain sufficient immunoprecipitated protein complexes for MS analysis, 6-8 dishes (10 cm) per experimental condition are required. As a control, the same number of dishes should be transfected with the empty expression vector.
Once the protein pellet is dissolved and volume is brought up to 1 mL, verify that the pH is approximately 7.5 by testing 5-pL aliquots on pH paper. Adjust with 1-5 pL of 1 M Tris-Base, pH 9, as required. Determine protein concentration using the Lowry assay and adjust to within 1-2 mg protein/mL. Dilute in MNT-S buffer if necessary.
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