Help in protein analysis
sai at intac.com
Mon Sep 26 21:38:45 EST 1994
The following has been posted by me on behalf of some people that do not
have newsgroup access. If some of you would be so kind as to give me some
information (or e-mail to cril at sangra.ncst.ernet.in) the scientists will
be very happy to receive the information. Please direct your responses to
the attention of Dr. Rajeswari Seshadri. (This is very important as the
whole institute has access only to this one e-mail address, and the
beauraucracy is such that unless a known person's name is visible, the
whole message is trashed!!)
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
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.
Dr. V. S. Lalitha M.D, Ph.D.
Head, Neuro Oncology Division,
Dr Rajeswari Seshadri Ph.D.
Scientific Officer - Neuro Oncology Division
c/o (either of the above)
Cancer Research Institute
Parel, Bombay 400012
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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