No specific band in GAL4 Western

Frederik Boernke boernke at IPK-Gatersleben.de
Fri Jun 13 12:50:24 EST 1997


>Hi Frederik
>
>One thing to be careful of is that last time i looked the Clontech 
>monoclonal GAL4 Ab didn't detect the fusion proteins produced from their 
>original 2 hybrid system, only the higher expression system 2. However, 
>things might have changed, and it sounds like you're using a more 
>sensitive chemiluminescent system anyway (and you might be using the 
>higher expression system). In which case, the one thing i'd say is you 
>seem to be diluting your antibodies an awful lot - i tend to use 1:1000 
>for my primary and 1:10000 for the secondary.
>Cheers,
>Joe Boutell
>
>
>
>boernke at IPK-Gatersleben.de (Frederik Boernke) wrote:
>>Hi!
>>
>>I am currently trying to detect the GAL4 fusion protein for my 2hybrid
>>screen. Therefor I use a Clontech monoclonal mouse Ab and Pierce
>>Super Signal Ultra Chemiluminescent Substrate
>>together with an Amersham biotin/streptavidin system.
>>The samples were prepared after the method of Printen and Spraque
>>(1994) and 20 ul were loaded onto a 15% mini gel. The primary Ab was
>>dilutet 1 : 10000, secondary 1: 100000 and the strepavidin-HRP also
>>1 : 100000. When I develop the blot the only thing I see is one major
>>band at about 50 - 50 kDa ( time of exposure: 10 sec). My question now
>>is, at which step should I try some optimization. Less Protein or higher
>>dilutions of Ab's. 
>>
>>Thanks in Advance,
>>Ricky
>>
>>
>>******************************************************************
>>
>>Frederik Boernke
>>Research Group Molecular Plant Physiologie
>>Institute for Plant Genetics and Crop Plant Research (IPK)
>>Corrensstr. 3
>>06466 Gatersleben
>>Tel.  039482 -5 321
>>Fax. 039482 -5 515
>>e-mail: boernke at ipk-gatersleben.de
>>http://www.ipk-gatersleben.de
>>
>
>
Hey Joe,

you seem to have some time writing e-mails, that's good!
I'm actually using a Stratagene expression system, which might 
express to low. About the chemilumenescence stuff: I phoned the 
tech service and they told me you can easily use dilutions of 1:1000000,
hard to believe, isn't (I didn't).
Do you think there is another way to verify expression of my fusion
protein or isn't really neccesarry, because I already sequenced the
construct and recognized the proper insert that way. Should I just
carry on screening the library?

Ricky

 

******************************************************************

Frederik Boernke
Research Group Molecular Plant Physiologie
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515
e-mail: boernke at ipk-gatersleben.de
http://www.ipk-gatersleben.de




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