Two-dimensional (2-D) gel electrophoresis consists of a first-dimension gel, which is IEF or isoelectric focusing gel, and second-dimension gel, or SDS-PAGE. IEF can separate proteins based on their isoelectric points (pI value); SDS-PAGE separates proteins according to their molecular weight. Generally, 2-D does not need to be performed and one-dimensional gel or SDS-PAGE can provide sufficient information for protein expression. However, a detailed characterization of a specific protein such as protein purification and sequencing may mandate 2-D gel electro-phoresis. The major disadvantage of one-dimensional gel electrophoresis using SDS-PAGE is that many proteins with the same molecular weight cannot separate. Instead, these proteins are shown in a single band in the gel. Normally, up to 30 to 40 bands can be seen in a single gel. In contrast, the major advantage of 2-D gel electrophoresis can provide incredible information about the pI values, molecular weight and species of proteins in a sample. If all goes well, hundreds of protein molecules can be revealed.

Detailed and simplified instructions for performance of IEF gel are available from the Immobilize DryStrip Kit (Pharmacia). The protocols for the second dimension gel or SDS-PAGE have been described previously.

Materials Needed


Sterile microcentrifuge tubes (0.5 or 1.5 ml)

Pipettes or pipetman (0 to 200 ml, 0 to 1000 ml)

Sterile pipette tips (0 to 200 ml, 0 to 1000 ml)

Gel casting plates

Gel combs

Electrophoresis apparatus

DC power supply Total protein samples Specific primary antibody Appropriate secondary antibody PVDF or nitrocellulose membranes 3MM Whatman filters

Multiphor II electrosemidry transfer apparatus (Pharmacia Biotech) or Hoe-

ffer TE 42 electroliquid blotting apparatus Shaker Plastic trays

Monomer Solution (30% Acrylamide) 116.8 g Acrylamide 3.2 g N,N-Methylene-to-acrylamide

Dissolve after each addition in 300 ml dd.H2O. Add dd.H2O to a final volume of 400 ml. Wrap the bottle with foil and store at 4°C. Caution: Acrylamide is neurotoxic. Gloves should be worn when working with this chemical.

Separating Gel Buffer 72.6 g 1.5 M Tris Dissolve well in 250 ml dd.H2O. Adjust pH to 8.8 with 6 N HCl. Add dd.H2O to 400 ml. Store at 4°C.

Stacking Gel Buffer

Dissolve well in 200 ml dd.H2O. Adjust pH to 6.8 with 6 N HCl. Add dd.H2O to 400 ml. Store at 4°C.

Dissolve well in 150 ml dd.H2O. Store at room temperature.

1% Ammonium Persulfate (AP) 0.2 g AP

Dissolve well in 2 ml dd.H2O. Store at 4°C for up to 5 days.

Overlay Buffer

25 ml Running gel buffer 1 ml 10% SDS solution

2X Denaturing Buffer

5 ml Stacking gel buffer

8 ml 10% SDS solution

4 ml Glycerol

2 ml b-Mercaptoethanol

Add dd.H2O to 20 ml.

0.02 g Bromophenol blue

Divide in aliquots and store at -20°C.

Running Buffer

12 g 0.25 M Tris 57.6 g Glycine 40 ml 10% SDS solution Add dd.H2O to 4 l.

1% (w/v) Coomassie Blue (CB) Solution 2 g Coomassie blue R-250

Dissolve well in 200 ml dd.H2O. Filter using 3MM Whatman paper.

Coomassie Blue Staining Solution 62.5 ml 1% CB solution 250 ml Methanol 50 ml Acetic acid Add dd.H2O to final 500 ml.

Rapid Destaining Solution 500 ml Methanol 100 ml Acetic acid Add dd.H2O to final 1000 ml.

Transfer Buffer

39 mM Glycine 48 mM Tris 0.04% SDS 20% Methanol

Phosphate Buffered Saline (PBS) 2.7 mM KCl 1.5 mM KH2PO4 136.9 mM NaCl 15 mM Na2HPO4 Adjust the pH to 7.2 to 7.4.

Blocking Solution

5% (w/v) Nonfat dry milk or 1 to 2% (w/v) gelatin or 2 to 3% (w/v) bovine serum albumin (BSA) in PBS solution. Allow the powder to be completely dissolved by shaking for 30 min prior to use.

Predetection/Color Developing Buffer 0.1 M Tris-HCl, pH 9.5 0.1 M NaCl 50 mM MgCl2

NBT Stock Solution

75 mg/ml Nitroblue tetrazolium (NBT) salt in 70% (v/v) dimethylformamide

BCIP Stock Solution

50 mg/ml 5-Bromo-4-chloro-3-indolyl phosphate (BCIP or X-phosphate), in 100% dimethylformamide


Chemiluninescent substrates Appropriate primary antibodies Appropriate secondary antibodies


1. Following electroblotting, the blotted membrane shows weak bands after Ponceau S staining, whereas the blotted gel displays clear bands stained with Coomassie blue. Obviously, the efficiency of protein transfer is very low. Try to set an appropriate transfer and to increase transfer time.

2. Protein bands are not sharp but are quite diffused. Current may be changed severely. Try to set up a constant power for electrophoresis.

3. No signal at all is evident. This is the worst situation in western blot hybridization and multiple factors may cause the problem.

a. Insufficient antigen (protein) was loaded into the gel. Try to increase the amount of proteins for each well.

b. Proteins may not be transferred at all onto the membrane. Make sure to stain the blotted membrane or use color protein markers as indicators.

c. The activity of primary or secondary antibodies, or both, is extremely low or does not work at all. Try to check the quality of antibodies by loading each in a separate well as controls in the same gel. If no signals are shown, it is most likely that western blot process has not been handled properly.

4. Hybridized signals are quite weak. The concentration of antigen is low or the activities of the antibodies may be low. To solve this problem, use appropriate concentrations of proteins or antibodies, increase exposure time for x-ray film or develop the membrane filter for a longer time.

5. A black background on x-ray film occurs when using chemilumines-cent detection. Multiple factors may be responsible for this common problem. The membrane filter may be dried out during antibody incubation or the washing process. Excess detection solution may not be wiped out prior to exposure. Exposure time may be allowed for too long. The solution is that the filter should be kept wet and excess detection reagents need to be wiped out. Try to reduce exposure time for x-ray film.

6. A highly purple/blue background is evident on the filter using colo-rimetric detection. Excess detection reagents may have been used or color developed too long. Try to apply an appropriate amount of detection reagents and pay close attention to color development. Once major bands become visible, stop development immediately by rinsing the filter with distilled water several times.

7. Nonspecific bands show up on x-ray film or membrane filter. This problem is most likely caused by the nonspecificity of antibodies. Make sure that the antibodies used are specific. One possibility is to increase blocking reagents and blocking time to block the nonspecific binding sites on the blotted membrane and elevate washing conditions using 0.05% Tween-20 in PBS.


1. Wu, W. and Welsh, M.J., Expression of the 25-kDa heat-shock protein (HSP27) correlates with resistance to the toxicity of cadmium chloride, mercuric chloride, cis-platinum(II)-diammine dichloride, or sodium arsenite in mouse embryonic stem cells transfected with sense or antisense HSP27 cDNA, Toxicol. Appl. Pharmacol., 141, 330, 1996.

2. Wu, W. and Welsh, M.J., A method for rapid staining and destaining of polyacrylamide gels, BioTechniques, 20, 3, 386-388, 1996.

3. Wu, W., Electrophoresis, blotting, and hybridization, In Handbook of Molecular and Cellular Methods in Biology and Medicine, Kaufman, P.B., Wu, W., Kim, D., and Leland, C., pp. 87-122, CRC Press, Boca Raton, FL, 1995.

4. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248-254, 1976.

5. Towbin, J., Staehlin, T., and Gordon, J, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76, 4350, 1979.

6. Knudsen, K.A., Proteins transferred to nitrocellulose for use as immunogens, Anal. Biochem., 147, 285, 1985.

7. Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, J.H., Improved method for utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose, Gene Anal. Technol., 1, 3, 1984.

8. Kyhse-Anderson, J., Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose, J. Biochem. Biophys. Methods, 10, 203, 1984.

Analysis of Cellular DNA or Abundance of mRNA by Radioactivity in Situ Hybridization (RISH)



Part A. Tissue Fixation, Embedding, Sectioning and Mounting of Sections on Slides

Preparation of Silane-Coated Glass Slides Method A. Silanization of Slides Method B. Treatment of Slides with Poly-L-Lysine Method C. Gelatin-Coating of Slides Preparation of Fixation Solutions

Preparation of 4% (w/v) Paraformaldehyde (PFA) Fixative (1 l) Fixation of Cultured Cells on Slides

Tissue Fixation, Embedding, Sectioning and Mounting of Sections on Slides

Fixation Embedding

Sectioning and Mounting Cryosectioning

Preparation of Frozen Specimens

Freeze Sectioning


Part B. In Situ Hybridization and Detection Using Isotopic Probes Dewaxing of Sections Protease Digestion

DNase Treatment for in Situ Hybridization of RNA RNase Treatment for in Situ Hybridization of DNA Preparation of Radioactive Probes

Synthesis of Probes for DNA Hybridization Using Random Primer Labeling of dsDNA

Preparation of [35S]UTP Riboprobe for RNA Hybridization by Transcription in Vitro Labeling

In Situ Hybridization Detection of Hybridized Signals Troubleshooting Guide References


Traditional methods for analysis of genomic DNA or mRNA expression include Southern blot or northern blot hybridization. In spite that the ability of Southern or northern blot hybridization to provide quantitative information on amounts of DNA or RNA in specific tissues, they cannot reveal spatial information on the distribution of DNA or RNA of interest within cells. Recently, a powerful and versatile technology has been developed to fulfill such a task: in situ hybridization.12 This technique becomes a very useful tool for scientists to simultaneously detect and localize specific DNA or RNA sequences with spatial information about their subcellular locations or locations within small subpopulations of cells in tissue samples. In situ hybridization can identify sites of gene expression, tissue distribution of mRNA, and identification and localization of viral infections.3-5

There are two major commonly used procedures for in situ hybridization and detection of cellular DNA or RNA.67 One is radioactivity in situ hybridization (RISH) using a radioactive probe6 and the other is fluorescence in situ hybridization (FISH), which utilizes nonradioactive probes.7 Each has its own strengths and weaknesses. Based on our experience, the RISH approach allows performing hybridizations at high temperatures (e.g., 65°C) and to employ washing under high-stringency conditions. As a result, a good signal-to-noise ratio can be obtained. However, the morphology of cells or tissues may not be well preserved.

In contrast, with the FISH approach, good images of cellular structure can be preserved, but this method has two major disadvantages. One is that with FISH the hybridization and washing procedures cannot easily be performed at high temperatures or under highly stringent conditions. The other limitation is that during final color development background cannot be readily controlled. As a result, nonspecific background may be a concern. The present chapter describes in detail the protocols for the success of in situ hybridization using isotopic probes. These methods have been well tested and have worked successfully in our laboratories.



High-quality glass slides play an essential role in preventing cells or tissue sections from falling off the slides during in situ hybridization. For this reason, regular glass slides should be precoated prior to use for in situ hybridization. Precoated glass slides are commercially available (e.g., Perkin-Elmer Corporation). Next, three commonly utilized methods that work well in our laboratories are described.

Method A. Silanization of Slides

1. Wash glass slides in 2 N HCl for 7 min using slide racks and glass staining dishes, and rinse the slides in dd.H2O for 2 x 1 min.

2. Rinse the slides in high-grade acetone for 1.5 min and air-dry.

3. Treat the slides in 2% organosilane solution (Aldrich Chemical Co.) diluted in high-grade acetone for 1.5 min with gentle agitation followed by rinsing in high-grade acetone for 1.5 min.

4. Air-dry the slides and store them in a box at room temperature until use. The slides can be stored up to 4 years.

Method B. Treatment of Slides with Poly-L-Lysine

1. Clean glass slides as described at step 1 and step 2 in Method A.

2. Immerse the slides in freshly prepared poly-L-lysine solution (Sigma, MW > 150,000, 1 mg/ml in dd.H2O) for 5 min.

Tip: Air bubbles should be avoided during submersion of slides.

3. Air-dry the slides and store at 4°C for up to 7 days.

Method C. Gelatin-Coating of Slides

1. Wash glass slides as described at step 1 and step 2 in Method A.

2. Coat the slides in 1% (w/v) gelatin in dd.H2O for 5 min at 4°C.

3. Air-dry the slides overnight at room temperature and store in a box at room temperature up to 4 weeks.

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