No specific band in GAL4 Western

Joe Boutell boutell at cf.ac.uk
Mon Jun 16 08:15:25 EST 1997


Hiya Ricky!

Well, i constructed a library using the Stratagene kit and from talking 
to their tech people i think this gives about the same level of 
expression as the original kit from Clontech, too low to detect with the 
GAL4 Ab. Having said that, a recent paper by Haydens group on a 
huntingtin interacting protein HIP1 (in Nature Genetics), used the pGBT9 
vector (low expression) and Y190 yeast strain and were able to Western 
blot for bait protein - you may want to look up exactly what they did. 
Personally, i just went ahead and screened and screened. If you don't get 
anything after a couple of screens of say >1 million clones, start 
thinking about other bait constructs (shorter regions, other regions of 
protein etc).
Cheers,
Joe Boutell    




boernke at IPK-Gatersleben.de (Frederik Boernke) wrote:
>>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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