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SantaCruz Antibody For Two Hybrid Assay (Again)

Shun'ichi Kuroda skuroda at inherit.biosig.kobe-u.ac.jp
Thu Feb 9 06:55:18 EST 1995

Dear Sirs:

I am an assistant professor of Kobe university.  Before the severve
earthquake on Jan 17, I posted same question to this NG.  But the 
RESs for my question could not be arrived at our server, since the 
server was broken by the earthquake.  So I repost the same question.

I am now trying to identify the PKC-binding protein by two hybrid assay.
Prior to screening, to identify probe protein (GAL4 DNA-binding domain 
fusion) in S.cerevisiae, I performed Western blotting analysis by using 
an antibody against GAL4 DBD (SantaCruz sc-577) .  
As previously posted (before earthquake), I obtained no specific band 
and several non-specidfic bands (30kDa and 90kDa) under the 1st 
antibody concentration of 1/1000.  After posting, the technical support 
staff of SantaCruz kindly and immediately advised me to increase the 
concentration of 1st antibody (1/100).  I repeated the Western blotting 
according to the suggestion.  However, I could not get an improved
data.  Even in the wild type S.cerevisiae, the band of wt GAL4 was not 
If anyone suceeded in identifying the probe protein by using the antibody,
please teach me the condition of Western blotting.  And, if anyone suceeded 
by using other type of antibody, please teach me the name of antibody and 
the condition.  
Any comments will be appreciated.
Many Thanks.

(For the SnataCruz staff)
Therefore, I want to get advise from the staff again.  Since my server is
broken by the earthquake, the name and address of the SantaCruz staff 
are unfortunately missed.  Please contact me.

E-mail: skuroda at inherit.biosig.kobe-u.ac.jp
Tel +81-78-803-1255   Fax +81-78-803-0994

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