APPENDIX E
Useful formulations for Western blotting and Immunoprecipitation

Cell Lysis Buffer

• 50 mM Tris-HCl, pH8.0

• 150 mM NaCl

• 1% NP-40 or Triton?X-100

2x Immunoprecipitation Buffer

• 2% Triton?X-100

• 300 mM NaCl

• 20 mM Tris, pH 7.4

• 1.0% NP-40

• 2 mM EDTA

• 2 mM EGTA

• 0.4 mM sodium vanadate (phosphatase inhibitor)

• 0.4 mM PMSF

2x SDS-PAGE Sample Buffer

• 100 mM Tris-HCl, pH 6.8

• 2% Sodium Dodecyl Sulfate (SDS)

• 20% Glycerol

• 0.2% Bromophenol Blue

• 2?0% b-mercaptoethanol (or DTT)

100x Protease Inhibitor Cocktail

• PMSF 5 mg (50 μg/mL)

• Aprotinin 100 μg (1.0 μg/mL)

• Leupeptin 100 μg (1.0 μg/mL)

• Pepstatin 100 μg (1.0 μg/mL)

• Add 100% Ethanol to 1 mL. Aliquot and store at -20癈.

APPENDIX F
Further Reading

General Background

1. Antibodies - A Laboratory Manual. 1996. Eds. E. Harlow and D. Lane. Cold Spring Harbor Laboratory.
2. Principles and Techniques of Practical Biochemistry, 4th edition. 1975. Wilson, K. and Walker, J., Cambridge University Press.
3. Monoclonal Antibodies: principles and practice, 3rd edition. 1996. Goding, J. W., Academic Press.

 Antigen Retrieval

1. Antigen retrieval techniques in Immunohistochemistry: Comparison of different methods. Pileri, S. et al. 1997. Journal of Pathology 183: 116-123.
2. Antigen retrieval in immunohistochemistry under the influence of pH using monoclonal antibodies. Shi, S.-R. et al. 1993. The Journal of Histochemistry and Cytochemistry 41: 1599-1604.
3. Microwave antigen retrieval for immunocytochemistry on formalin-fixed, paraffin-embedded post-mortem CNS tissue. McQuaid, S. et al. 1995. Journal of Pathology 176: 207-216.

Immunohistochemistry and Immunocytochemistry

1. Immunocytochemistry: Modern methods and applications. Polak, J. M and Van Noorden, S. (eds). 1986. Bristol: Wright.
2. Advances in Immunohistochemistry. Hsu, S.-M. and Raine, L. 1984. DeLellis, R. A. (ed.). New York: Masson 31-42.

Immunochemistry and Immunochemical Techniques

1. Immunology: A Short Course, 2nd edition. Benjamini, E. and Leskowitz, S. 1991. Wiley-Liss New York.
2. Fundamental Immunology, 2nd edition. Coleman, R. M., Lombard, M. F. and Sicard, R. E. 1992. W. C. Brown, Dubeque, I. A..
3. Practical Immunology, 3rd edition. Hudson, L. and Hay, F. C.. 1989. Blackwell Scientific Publications, Oxford.
4. Essential Immunology, 7th edition. Roit, S. M. 1991. Blackwell Scientific Publications, Oxford.
5. Monoclonal Antibodies: principles and practice, 3rd edition. 1996. Goding, J. W., Academic Press.
6. Antibody Affinity: Thermodynamic Aspects and Biological Significance. Steward, M. W. and Steensgaard, J. 1983. Boca Raton: CRC Press.
7. Immunocytochemistry, 2nd edition. Stermberger, L. A. 1979. New York: Wiley.
8. Multispecific monoclonal antibodies. Ghosh, S. and Campbell, A. M. 1986. Immunology Today 7:217-222
9. Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications. Andrews, A. T. 1986. Oxford University Press, Oxford.
10. Gel Electrophoresis: Proteins. Dunn, M. J. 1993. Bias Scientific, Oxford.
11. Hjelmeland, J. M. and Chrambach, A. (1984). Solubilization of functional membrane proteins. Methods Enzymol. 104:305?18.
12. Welling, G. W. et al. (1987). Column liquid chromatography of integral membrane proteins. Chromatography 418:223?43.
13. Mozdzanowski, J. et al. (1992). High yield electroblotting onto polyvinylidine difluoride membranes from polyacrylamide gels. Electrophoresis 13:59?4.