Characterisation of a Polyclonal Antiserum  Againts Ubiquitin and its use in the Investigation  of Ubiquitin Conjugate Distribution in Normal  and Stressed 3T3-LI Cells

İbrahim PİRİM

Makale detayları

Characterisation of a Polyclonal Antiserum Againts Ubiquitin and its use in the Investigation of Ubiquitin Conjugate Distribution in Normal and Stressed 3T3-LI Cells

Yazarlar:

İbrahim PİRİM

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Medical Sciences 1998 Vol.28 Issue 6, pp.583-590

Belge Tipi:

Makale

Yayın Tarihi:

5/1998

Belge Dili:

Türkçe

Document ID (Org No.):

2000758154

Sıklık:

İki Aylık

eISSN:

1303-6165

pISSN:

1300-0144

Anahtar kelimeler:

Özet:

 

Antiserum against ubiquitin was  used to identify ubiquitin conjugates in 3T3-  LI cells, and to investigate changes in the  amount and distribution of these conjugates  in cells subjected to stress. Stress was  induced by incubation with canavanine, an  amino acid analogue, and by exposure of cells  to temperatures of 43°C (heat shock).  SDS-PAGE, followed by immunoblotting,  produced no observable change in ubiquitin  conjugation with the canavanine as used in  this protocol.Heat shock, in a variety of  different exposure protocols, appeared to  produce an increase in a wide spectrum of  ubiquitin conjugates of molecular  weight>70kd.  The conjugates wereresent in the Triton-extracted  cell residues, which are nuclear-cytoskeletal-  enriched preparations and this  fact is discussed in the light of evidence for a  site for protein sequestration prior to  degradation which localises to a nuclear-intermediate  filament-enriched cell fraction.  Indirect immunofluorescence was employed  in an attempt to visualize changes in the  distribution of ubiquitin in whole cells which  had been exposed to stress. No changes were  observed, and this is discussed in terms of  the specificity of the antiserum for different  forms of ubiquitin, and in the light of other  work which was successfully used the  antiserum to visualize ubiquitin associated  with intracellular inclusions.  Experiments are proposed to investigate  more fully the changes in ubiquitin  conwugation and their likely meaning with  respect to protein degradation. Modification  of procedures for microscopy are proposed,  in order to visualize changes in ubiquitin  distribution in whole cells, and to assess  whether any such changes have relevance to  pathological processes involving ubiquitin.

 

Antiserum against ubiquitin was  used to identify ubiquitin conjugates in 3T3-  LI cells, and to investigate changes in the  amount and distribution of these conjugates  in cells subjected to stress. Stress was  induced by incubation with canavanine, an  amino acid analogue, and by exposure of cells  to temperatures of 43°C (heat shock).  SDS-PAGE, followed by immunoblotting,  produced no observable change in ubiquitin  conjugation with the canavanine as used in  this protocol.Heat shock, in a variety of  different exposure protocols, appeared to  produce an increase in a wide spectrum of  ubiquitin conjugates of molecular  weight>70kd.  The conjugates wereresent in the Triton-extracted  cell residues, which are nuclear-cytoskeletal-  enriched preparations and this  fact is discussed in the light of evidence for a  site for protein sequestration prior to  degradation which localises to a nuclear-intermediate  filament-enriched cell fraction.  Indirect immunofluorescence was employed  in an attempt to visualize changes in the  distribution of ubiquitin in whole cells which  had been exposed to stress. No changes were  observed, and this is discussed in terms of  the specificity of the antiserum for different  forms of ubiquitin, and in the light of other  work which was successfully used the  antiserum to visualize ubiquitin associated  with intracellular inclusions.  Experiments are proposed to investigate  more fully the changes in ubiquitin  conwugation and their likely meaning with  respect to protein degradation. Modification  of procedures for microscopy are proposed,  in order to visualize changes in ubiquitin  distribution in whole cells, and to assess  whether any such changes have relevance to  pathological processes involving ubiquitin.

Makale detayları

Characterisation of a Polyclonal Antiserum Againts Ubiquitin and its use in the Investigation of Ubiquitin Conjugate Distribution in Normal and Stressed 3T3-LI Cells

Yazarlar:

İbrahim PİRİM

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Medical Sciences 1998 Vol.28 Issue 6, pp.583-590

Belge Tipi:

Makale

Yayın Tarihi:

5/1998

Belge Dili:

Türkçe

Document ID (Org No.):

2000758154

Sıklık:

İki Aylık

eISSN:

1303-6165

pISSN:

1300-0144

Ubiquitin

Heat Shock

Stress.

Sayı

Makale detayları

Characterisation of a Polyclonal Antiserum Againts Ubiquitin and its use in the Investigation of Ubiquitin Conjugate Distribution in Normal and Stressed 3T3-LI Cells

Yazarlar: İbrahim PİRİM

Yayıncı: TUBITAK

Dergi: Turkish Journal of Medical Sciences 1998 Vol.28 Issue 6, pp.583-590

Belge Tipi: Makale

Yayın Tarihi: 5/1998

Belge Dili: Türkçe

Document ID (Org No.): 2000758154

Sıklık: İki Aylık

eISSN: 1303-6165

pISSN: 1300-0144

Ubiquitin

Heat Shock

Stress.

Sayı

Yazarlar:

İbrahim PİRİM

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Medical Sciences 1998 Vol.28 Issue 6, pp.583-590

Belge Tipi:

Makale

Yayın Tarihi:

5/1998

Belge Dili:

Türkçe

Document ID (Org No.):

2000758154

Sıklık:

İki Aylık

eISSN:

1303-6165

pISSN:

1300-0144