Blotting from Immobilized pH Gradient Gels: Application to Total Cell Lysates

Summary

Isoelectric focusing as used in the first dimension of two-dimensional gel electrophoresis separates protein isoforms such as those due to phosphorylation and acetylation. The immunoblotting method described here reveals this diversity by a one-dimensional separation. Using commercially available immobilized pH gradient plates or strips, the resolved proteins are transferred to PVDF membranes by diffusion and are probed with protein specific antibody. The system is useful for monitoring changes of banding patterns and permits parallel processing of samples. Since the effect of posttranslational modifications on the isoelectric point can be predicted, inferring the number and extent of modifications is possible.

1 Introduction

When looked at closely, proteins both intracellular and secreted, display a bewildering number of modifications. Because of the importance of posttranslational modifications for regulating biological activities of proteins, there is a corresponding effort aiming at detecting and monitoring their occurrence. An increasing number of antibodies are becoming available that allow specific detection of modifications on selected proteins, mostly well characterized components of signaling cascades. Such antibodies are, however, often not available for other proteins. The method described in this chapter can be used to detect and track protein modifications that lead to alterations of their charge and consequently of a change of the isoelectric point. Such charge isoforms can be separated by isoelectric focusing and are evident from two-dimensional separations where trains of spots are seen that are frequently due to different phosphorylation states. Especially high resolution is achieved by IEF on immobilized pH gradient gels. Although SDS-PAGE is sometimes capable of resolving modified proteins, the extent or even direction of the changed migration is difficult to predict.

The IEF blotting method described here was developed for monitoring alterations occurring on intracellular proteins that require denaturing conditions for extraction and separation (1). The method allows sensitive detection and estimation of the relative abundance of isoforms with antibodies. The latter is a quantity that is not easily measured by modification specific reagents. Quantification is facilitated by running samples side by side under the same conditions. In principle, similar information can be obtained from two-dimensional separations by running a series of westerns. Results from such experiments are, however, difficult to quantify because of unavoidable technical variability. The separation method used here is very similar to the first dimension of two-dimensional separation systems (2, 3). It uses commercially available precast gels and allows direct comparison with two-dimensional separations. Transfer from the plastic supported gels is achieved by diffusion blotting.

2 Materials

• 1. IPG plates (immobilized pH gradient gels) Immobiline DryPlates (GE Healthcare or other manufacturers) or immobilized pH gradient strips (see Note 1). Various pH ranges are available.

• 2. R-Buffer (4): 4% CHAPS, 7 M urea, 2 M thiourea, 10 mg/mL dithiothreitol, and 1% carrier ampholytes (pH 3–10) (Pharmalytes; Amersham Pharmacia Biotech, Uppsala, Sweden), 0.02 mg/mL bromophenol blue. Store at −80°C.

• 3. Reswelling buffer: R-buffer without carrier ampholytes and without bromophenol blue. Store at −80°C.

• 4. TCA fixative: 12% trichloroacetic acid, 3.5% sulfosalicylic acid.

• 5. Multiphor II Electrophoresis System (GE Healthcare).

• 6. Sample applicator for IEF (see Note 2).

• 7. Transfer buffer: 4 M guanidinium chloride, 0.05 M Tris-HCl pH 8, 1 mg/mL DTT (added shortly before use).

• 8. Filter paper e.g. Whatman 1Chr, Cat. No. 3001917.

• 9. Polyvinylidene difluoride (PVDF) membrane.

3 Methods

3.1 Preparation of Extracts

• 1. Collect cells by centrifugation, wash once with PBS, suspend the pellet, transfer into a tared microcentrifuge tube, centrifuge once more and remove as much of the supernate as possible (see Note 3). Estimate the volume by weighing and add R-buffer corresponding to five volumes of the cell pellet, suspend and leave on ice for about 1 h. Centrifuge at 1,000 g for 15 min in the cold. Store the sample at −80°C. Protein concentration may be determined by a Coomassie binding assay (5) (see Note 4). Protein concentrations are in the range of 3–10 mg/mL.

3.2 Isoelectric Focusing on IEF Immobiline Plates

• 1. Reswelling polyacrylamide sheets in R-buffer devoid of ampholines (see Note 5): The procedure is otherwise according to instructions of the manufacturer. Cut required size if only part of a sheet is used, and mark the polarity of the pH-gradient by cutting off a corner from the sheet. Place the volume of buffer required for the sheet in the reswelling tray and place the sheet face down on the liquid. Avoid trapping air bubbles and spilling of buffer over the top side of the sheet. Leave overnight at room temperature in a box containing wet paper towels for achieving a water-saturated atmosphere. Instead of Immobiline plates, it is also possible to use individual IEF strips (see Note 1).

• 2. Place about 2 mL of kerosene on the cooling stage of the Multiphor apparatus and position the IPG plate paying attention to match the IPG plate to the required polarity of the electrodes. Remove excess buffer with a filter paper (provided for that purpose by the manufacturer). Position electrode filter paper strips and sample applicator on the gel surface (about 2 cm from the anode). The position may vary according to the proteins of interest.

• 3. Using a micropipette apply the samples (1 µL or up to 4 µL for the larger sample comb), which will be held by capillary force underneath the indentations (see Note 6).

• 4. Running conditions: 15°C, from 0 to 300 V in 30 min, then to 900 V in 9 h, keeping at 900 V for 6 h, raising to 3,500 V within 1 h, keep at 3,500 V for a final 4 h. The entire run takes 20 h with an accumulated Volt-hour product of ∼27,000 Vh. We avoided focusing at the highest voltages without attention. Adhere to the safety instructions of the IEF manufacturer. Depending on the pH range and type of protein, different focusing conditions may be needed.

3.3 Transfer to PVDF Membrane

• 1. Incubate the gel with TCA fixative for 1 h. Wash three times with water, 10 min each (see Note 7).

• 2. Cut a piece of PVDF to the size of the gel, moisten it by soaking in ethanol followed by washing in water.

• 3. Prepare 10 pieces of filter paper (e.g., Whatman 1Chr, Cat. No. 3001917), 1 cm larger than the membrane, and soak in transfer buffer (about 35 mL is required for half a gel, ∼13 × 13 cm²).

• 4. Assemble the sandwich by placing the gel on a glass plate, followed by the PVDF membrane, and a stack of filter papers soaked in the transfer buffer. Overall thickness of the paper stack will be about 2 mm. Wrap the assembly with a plastic foil to prevent drying out and cover with glass plate (Fig. 1). Ensure good contact by compressing with a heavy object and leave overnight at room temperature.

• 5. Wash the membrane with water to remove guanidinium chloride.

• 6. At this stage, the membrane is processed like any western blot.

Fig. 1

Setup used for diffusion transfer from plastic backed Immobiline DryPlates.

3.4 Visualization of Total Protein Pattern (Optional)

To assess any disturbances or differences between samples, we find it useful to visualize and photograph the total protein pattern on the PVDF membrane using stains that are compatible with subsequent immunostaining (e.g., SyproRuby). Also, if needed, IEF standards may be added in adjacent lanes and thus be visualized on the membrane.

3.5 Interpretation

It is useful to judge whether an assumed modification can account for the observed shift of the isoelectric point of the protein under investigation. Thus, for proteins with known amino acid composition, the isoelectric point can be predicted (6). The effect of phosphorylation can also be calculated (7). For acetylation on lysine groups, it is possible to simply omit one lysine from the sequence before computation of the pI. Similarly, other modifi-cations may be incorporated by considering their effect on the charge of the protein. Figure 2shows an example of an application of the IEF blotting method.

Fig. 2

Example of an IEF blotting experiment with extracts from rat peripheral blood white blood cells. The protein extract loaded (4 µL) corresponds to cells obtained from 80 µL of blood. The separation was carried out on Immobiline plates pH range 4.5–5.4. Transfer to PVDF membranes was achieved by diffusion blotting. An antibody against 14-3-3gamma protein detects two bands in untreated animals and three bands in animals that had been treated with bengamide. This compound inhibits methionine aminopeptidase (8). As a consequence, acetylation of the penultimate N-terminal aminoacid is also inhibited and a slightly more basic protein accumulates. The position of normal acetylated protein is marked as “a,” the unprocessed form as “b.” The nature of the intermediate form is not known. A: untreated; B, C, and D: animals treated with increasing doses of bengamide.

4 Notes

• 1. Instead of Immobiline DryPlates, individual strips may also be used. Here, the procedures for the first dimension of 2D electrophoresis are closely adhered to. Thus, reswelling of the gel strips proceeds in the presence of the sample, thereby allowing application of higher sample volumes. After electrophoresis, the strips are processed exactly as described for the plates. For the blotting step, the strips can be arranged side by side. A large choice of strips covering wide and narrow pH ranges is commercially available. For strips, an electrophoretic transfer method, which requires removal of plastic backings, has been described (9).

• 2. The applicator comb may be built by cutting a disposable applicator intended for the Phast Gel™ system (PhastGel™ Sample Applicators, 8× of 1 µL or 6× of 4 µL, Cat. No. 18-1618-01 or 18-0012-29, respectively). The comb needs to be cut horizontally to fit to the limited height available on the Multiphor II. A rectangular piece of the plastic material is glued to one end of the comb with a drop of dichloromethane (this solvent is toxic and irritant, work under a fume hood) (see Fig. 3).

• 3. It is important to keep the salt concentration as low as possible as high conductivity leads to disturbances during running.

• 4. We find it convenient to carry the assay out in 96-well microtiter plate by adding 0.5 and 1 µL of the sample to 10 µL water and then adding 150 µL prediluted Bradford reagent to the wells. This amount of R-buffer is tolerated in this assay.

• 5. We found that ampholines at the relatively high concentrations used for focusing as well as CHAPS have a tendency to bind to the blotting membrane. Hydrophilic proteins may poorly bind in the presence of these components.

• 6. Other methods of sample application may also be useful, such as cup loading or paper pieces.

• 7. This step serves to wash out CHAPS, which interferes with adsorption of many proteins. Large or hydrophobic proteins may not require the fixation and CHAPS washout step, one example being tubulins (1). If IEF blotting is to be carried out repeatedly, it is worthwhile to test simplified transfer procedures.

Fig. 3

T-shaped sample applicator.