This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

CYTOSKELETON
Carla Fiorentini*, Paola Matarrese, Alessia Fabbri and Walter Malorni
Department of Ultrastructures, Istituto Superiore di Sanità, Viale Regina Elena 299, OO161 Rome, Italy
*Corresponding author: fax: +39-6-49387140; email: fiorentini@ul.net.iss.it

 
Introduction



   

 
Fluorescence microscopy


 
A.1 Introduction

A.2 Protocol  A.2.1 Materials
   

A.2.2 Methods

 
Standard protocol

Perform steps from 1 to 3 only for floating cells. For cells grown on coverslips consider step 4 as the first.

 

  1.  Centrifuge cells at 600 rpm for 5 min.
  2.  Resuspend the pellet in 40 µl of PBS (see Appendix A1).
  3.  Deposit 30 µl of cell suspension on a coverslips coated with poly-L-lysine (as reported in Appendix 1) for 20 min.
  4.  Rinse rapidly the cells adhering to the coverslip with medium devoid of serum.
  5.  Fix cells with paraformaldehyde solution (see Appendix 1) for 10 min.
  6.  Wash cells 3 times with PBS buffer.
  7.  Permeabilize cells with Triton X-100 (see Appendix A1) for 5 min.
  8.  Preincubate cells with PBS 1% heat-inactivated FCS or BSA, for 15 min to prevent unspecific immunoglobulin adsorption.
  9. Incubate the cells with specific antibody for 30 min at 37°C.
  10. Wash cells 3 times with PBS buffer.
  11. For indirect method, incubate the cells with labelled second antibody for 30 min at room temperature. For direct method go directly to step 13.
  12. Wash cells 3 times with PBS buffer.
  13. Mount the coverslips with glycerol-PBS (2:1).
  14. Analyze with a fluorescence microscope.
 
 Protocol advised for cytokeratins' detection
   

Protocol advised for polymerized actin (F-actin) detection

Protocol advised for antigens easily degradable
   

 
A3. Commentary



 

A3.1. Background Information

A3.2. Critical Parameters 1. The affinity of the antibody for the antigen. Polyclonal or monoclonal antibodies can be used for detection of cytoskeletal proteins, although the use of polyclonal antibodies also causes higher non-specific backgrounds.

2. The choice of an appropriate experimental protocol for detecting the cytoskeletal proteins of interest.
 

A3.3. Troubleshooting
  A3.4. Anticipated Results A3.5. Time Considerations Appendix A1

Phosphate Buffer Solution (PBS) 1x
 

 
Paraformaldehyde fixative solution  
Triton permeating solution  Saponine permeating solution Nonidet permeating solution  
Poly-L-lysine solution  

For preparation of coated coverslips: deposit 40 m l of this solution on 13 mm coverslips and wait the complete evaporation. Store the coverslips at room temperature protecting them from the dust.

 
Appendix A2
 

Appendix A3  
 
B) Flow cytometry
 
B1. Introduction

The quantification of cytoskeletal proteins can be obtained by using flow cytometry. Measurements of fluorescent dyes bound to cellular constituents represent the majority of flow cytometric applications. Fluorescence has three major advantages in flow studies: i) fluorescence emission is direcltly proportional to specific cell constituents; ii) low concentration of dye per cell can be detected; and iii) nonfluorescent compounds can be made fluorescent by intracellular enzymes.

B2. Protocol
 

B2.1. Materials

B2.2. Methods

 

Standard protocol for cytoskeletal proteins' detection by flow cytometry
 

  1. Wash the cell suspension with PBS
  2. Centrifuge cells at 800 rpm for 5 min.
  3. Resuspend 106 cells in 1 ml of PBS (see Appendix B1).
  4. Add 2 ml of paraformaldehyde solution (see Appendix B1) for 10 min at room temperature.
  5. Wash cells twice with PBS buffer.
  6. Centrifuge cells at 800 rpm for 5 min.
  7. Resuspend cells in 1 ml of cold PBS.
  8. Add 1 ml of cold (-20°C) methyl alcohol
  9. Incubate 10 min at 4°C.
  10. Wash cells twice with PBS buffer.
  11. Resuspend cells in 50 m l PBS-Nonidet P-40 0.5 % (see Appendix B1).
  12. Incubate cells with specific antibody for 1 hour at room temperature.
  13. Wash cells twice with PBS buffer.
  14. For the indirect method, incubate the cells with labelled second antibody for 30 min at room temperature. For the direct method go directly to step 15.
  15.  Wash cells twice with PBS buffer.
  16.  Resuspend cells in 500 µl of cold PBS.
  17.  Analyze with a cytofluorimeter.
 
Protocol advised for antigens easily degradable

Repeat steps from 1 to 4 of the standard protocol.

 

5. Add 2 ml of paraformaldehyde solution (see Appendix B1) for 10 min at 4°C.

6. Add 200 m l of Triton solution (see Appendix B1) for 10 min at 4°C.

7. Wash cells twice with cold PBS buffer.

8. Centrifuge cells at 800 rpm for 5 min.

9. Resuspend cells in 1 ml of 1% AB human serum in cold PBS.

10. Wash cells with PBS buffer.

11. Resuspend cells in 50 m l of 1% AB human serum in cold (see Appendix B1).

12. Incubate cells with specific antibody for 1 hour at 4°C.

13. Wash cells twice with PBS buffer.

14. For the indirect method, incubate the cells with labelled second antibody for 30 min at 4°C. For the direct method go directly to step 16.

15. Wash cells twice with PBS.

16. Resuspend cells in 500 m l of cold PBS.

17. Analyze with a cytofluorimeter.

 
 

 B3. Commentary

 

B3.1. Background Information
See A3.1

 

B3.2. Critical Parameters
See A3.2.

 

B3.3. Troubleshooting

B3.4. Anticipated Results B3.5. Time Considerations
   Appendix B1

 Phosphate Buffer Solution (PBS) 1x

EDTA solution
  Trypsin solution
  Paraformaldehyde fixative solution Triton permeating solution
   Nonidet permeating solution
   

Appendix B2

Appendix B3
 
C) Immunoprecipitation

 
C1. Introduction C2. Protocol C2.1. Materials C2.2. Methods

 

Lysis of cells and preclearing lysate

 

1. Wash cells with PBS at room temperature. Drain well.

2. Incubate cells in lysis buffer A (107 cells in 1 ml buffer prechilled to 4°C) for 1 h at 4°C.

3. Scrape the cells and debris from the plate with a rubber policeman and transfer all the preparation to a 1.5 ml conical tube.

4. Centrifuge 10 min at 10,000 g at 4°C.

5. (Optional) Centrifuge the supernatant at 100,000 g for 30 min.

6. Quantify the protein concentration and use the same amount of proteins for all samples.

7. Preclear supernatant by adding 20 m l of control Protein A/G agarose prepared without antibody or coupled to non specific antibody.

8. Shake on an orbital shaker for 1 h at 4°C.

9. Centrifuge 1 min at 10,000 g and save the supernatant.

(Optional from 7 to 9)

 

Forming the antibody-antigen complex Purification of the immune complex 12. Add 20 m l of Protein A/G plus Agarose and incubate on an orbital shaker at 4°C for 1 h.

13. Centrifuge at 10,000 g for 1 min at 4°C.

14. Remove the supernatant by aspiration and add 0.5 ml of lysis buffer. Vortex to resuspend.

15. Wash three times with lysis buffer. Remove the last wash as completely as possible.

16. Add 20 m l of Leammli sample buffer 1X. Heat for 5 min at 100°C, centrifuge at full speed and use the released immune complex for SDS-polyacrilamide gel electrophoresis (SDS-PAGE) (5).

17. Detection of the immunoprecipitation reaction can be performed with the following methods:

a) Comassie Brilliant Blue or Silver staining (ref 5) if the amount of protein is >1 m g.

b) Transfer to nitrocellulose and blotting (ref 5) if the amount of proteins is <1 m g.

c) Autoradiography (ref 6) if you labelled with radioactive amino acid to detect very rare proteins

 
 

C3. Commentary

 

C3.1. Background information

C3.2. Critical parameters 1. As mentioned above, the affinity of the antibody for the antigen is one of the critical parameters for good resolution in immunoprecipitation. This gives the strength of the binding of an epitope to an antibody and since immunoprecipitation relies on the formation of antigen-antibody complex in solution at relatively low concentration of the antigen, affinity of at least 108 mol-1 are required. Polyclonal antibodies are the most commonly used for immunoprecipitation. They contain antibody that bind to multiple sites on the antigen, thus providing a high affinity for the antigen and a more stable antigen-antibody-protein A complex. Although using polyclonal antibodies for immunoprecipitation often produce stable multivalent interactions, their use also causes higher non-specific backgrounds. Monoclonal antibodies bind to only one epitope thus providing a specific tool to identify a particular structure on an antigen. In addition, the immune complexes formed using monoclonal antibodies give less of a problem with non-specific binding, the backgrounds, in general, being cleaner. Although monoclonal antibodies may solve problems of background, their use also creates some difficulties. The major problem is given by the affinity. Because the antigen is held, in general, by one antibody-antigen interaction, monoclonal antibodies with affinities lower than 107 mol-1 are difficult to use in immunoprecipitation. Another problem by using monoclonal antibodies is the possibility of detecting cross-reactions with other polypeptides. An epitope, in fact, can be a relatively small structure (5-7 aminoacids), thus there is a reasonable chance that a similar epitope can be found on another polypeptide.

 

2. In general, the conditions used for lysis should be as gentle as possible to retain the antibody binding site and to avoid solubilizing background proteins, but they have to ensure quantitative release of the antigen. Salt concentration, type of detergent, presence of divalent cations and pH are all variables that affect the release of polypeptide antigens. The following parameters should be monitored to determine the optimal conditions of extraction:
 

- salt concentrations between 0 and 1 M

- non ionic detergent concentrations between 0.1 and 2%

- ionic detergent concentrations between 0.01 and 0.5%

- divalent cations concentrations between 0 and 10 mM

- pH between 6 and 9
 

 

3. A gel run after immunoprecipitation contains not only the protein of interest, but also the antibodies as one of the major band. Antibodies are formed by light and heavy chain which together have a molecular weight of about 150 kDa. The light chain alone is at the MW of many important proteins which are usually studied by immunoprecipitation. Thus, in the case of Comassie blue, silver staining and immunoblotting is recommended to use a Laemmli buffer without DTT so that the two chains are not separated during migration and they can be found as a unique band at almost 150 kDa. Also, to avoid this problem in immunoblotting, different antibodies might be used to immunoprecipitate and to detect the protein after transfer, i.e. polyclonal antibodies to immunoprecipitate and monoclonal antibodies to detect the protein. Thus, the secondary antibody will be an anti-mouse which will not recognize the polyclonal immunoglobulin used to immunoprecipitate.

 

C3.3. Troubleshooting
  C3.4. Anticipated results C3.5. Time considerations Appendix C1
 

Phosphate Buffer Solution (PBS) 1x

Lysis buffer A Leammli buffer 1(x) 
   
Comassie Brilliant Blue
  Appendix C2 Appendix C3

1. Ultracentrifuge

 

Key References

 

1. Meredith, J.E. Jr, and M.A. Schwartz. 1997. Integrins, adhesion and apoptosis. Trends Cell Biol. 7, 146-150.

2. Sambrook, J., E.F. Fritsch, T. Maniatis. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, eds.

3. Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389-399.

4. Immunocytochemical technology. G. V. Childs, ed. Alan R. Liss, Inc., New York.

5. Harlow, E., D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, eds.

6. Coons, A.H., and M.H. Kaplan. 1950. Localization of antigens in tissue cells. Improvements in a method for the detection of antigen by means of fluorescent antibody. Journal Experimental Medicine 91: 1-13.

7. Wary, K.K, F. Maniero, S.J. Isakoff, E.E. Marcantonio, and F.G. Giancotti. 1996. The adaptor protein shc couples a class of integrins to the control of cell cycle progression. Cell 87 (4): 733-742.

8. Defilippi, P., M. Venturino, D. Gulino, A. Duperray, P. Boquet, C. Fiorentini, G. Volpe, M. Palmieri, L. Silengo, and G. Tarone. 1997. Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. J. Biol. Chem. 272: 21726-21734.

9. Rouslahti, E. 1997. Stretching is good for a cell. Science 276, 1345-1346.

10. Fiorentini, C., A. Fabbri, P. Matarrese, L. Falzano, P. Boquet, and W. Malorni. 1997. Hinderance of apoptosis and phagocytic behaviour induced by E. coli cytotoxic necrotizing factor 1 (CNF1): two related activities in epithelial cells. Biophys. Biochem. Res. Com. 241:341-346.

11. Haldar, S., A. Basu, and C.M. Croce. 1997. Bcl2 is the guardian of microtubule integrity. Cancer Res. 15: 229-233.

 

 
FIGURE LEGENDS