Help in microsequencing & homology detection

Seshadri Narasimhan sai at intac.com
Sat Sep 10 14:32:11 EST 1994


Hi there to everyone in cyber-space!!
I am posting the following information from Cancer Research Institute 
(Bombay, INDIA). Please e-mail responses to:

		cril at sangra.ncst.ernet.in
		Attention: Dr. Rajeswari Seshadri

______________________________________________________________________________
Teratocarcinoma-derived neurotrophic factor:

We have developed a translatable teratocarcinoma model in C3HJax mice in our
lab. This type of tumor, which is equivalent to a developing embryo, is known
to produce various growth promoting factors. During our search for such a
factor which would promote growth of neural elements, employing the standard
method for isolation of Nerve growth factor (Mr 26,000), we observed three (3) 
bands with Mr 50kD, 60kD and 72kD on SDS-PAGE. All these bands were eluted 
individually from the gel and their biological activity was tested on
cerebellar explant cultures from newborn rats. The neurite extension and 
proliferation of neuronal cells was maximum in the 60kD protein and we decided 
to focus our attention on it for further studies. At present we are in the 
process of producing polyclonal antibody against this 60kD protein with the 
aim of immunohistochemically localizing it in a variety of normal & cancerous 
nervous tiisues.

The lypophilised powder is available for 1) microsequencing off proteins to 
as much length of amino acids as possible, 2) comparison of this sequence 
with already known protein sequences to detect any homology.

We would like someone to help us due to our lack of expertise/experience in 
microsequencing. Please contact us either via e-mail or snail-mail. (The 
attached file gives additional information regarding the protocol).
-------------- next part --------------
Teratocarcinoma-derived neurotrophic factor:

We have developed a translatable teratocarcinoma model in C3HJax mice in our
lab. This type of tumor, which is equivalent to a developing embryo, is known
to produce various growth promoting factors. During our search for such a
factor which would promote growth of neural elements, employing the standard
method for isolation of Nerve growth factor (Mr 26,000), we observed three (3) 
bands with Mr 50kD, 60kD and 72kD on SDS-PAGE. All these bands were eluted 
individually from the gel and their biological activity was tested on
cerebellar explant cultures from newborn rats. The neurite extension and 
proliferation of neuronal cells was maximum in the 60kD protein and we decided 
to focus our attention on it for further studies. At present we are in the 
process of producing polyclonal antibody against this 60kD protein with the 
aim of immunohistochemically localizing it in a variety of normal & cancerous 
nervous tiisues.

Protocol for isolation of 60kD protein:
1) 20 g tumor homogenized in 100 ml distilled water.
2) Centrifuge at 14500 rpm for 1 hr.
3) To the supernatent 1/9 volume of 0.5M acetate buffer (pH 4.0) and solid NaCl
   wa added to bring the final concn. of NaCl to 0.4M
4) Centrifuge at 14500 rpm for 30 mins.
5) Supernatent loaded onto a carboxy methyl cellulose column equilibrated with
   0.05M acetate buffer with 0.4M NaCl (pH 4.0)
6) Non-adsorbing material washed with same buffer
7) Column washed with 0.05M acetate buffer (pH 4.0) (till absorbance at 280 nm 
   is less than 0.001)
8) Final elution with Tris-HCl buffer with 0.4M NaCl (pH 9.0)

The final eluate is subjected to SDS-PAGE. Proteins are eluted in ammonium 
bicarbonate SDS buffer after KCl visualization and lypophilised.

The lypophilised powder is available for 1) microsequencing off proteins to 
as much length of amino acids as possible, 2) comparison of this sequence 
with already known protein sequences to detect any homology.

Principal investigator:
Dr. V. S. Lalitha M.D, Ph.D.
Head, Neuro Oncology Division,

Co-investigator:
Dr Rajeswari Seshadri Ph.D.
Scientific Officer - Neuro Oncology Division

Postal address:
c/o (either of the above)
Cancer Research Institute
Parel, Bombay 400012
INDIA

e-mail address:
cril at sangra.ncst.ernet.in


We would like someone to help us due to our lack of expertise/experience in 
microsequencing. Please contact us either via e-mail or snail-mail.


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