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non radioactive Dig-EMSA-please help


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#1 rgn

rgn

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Posted 30 January 2011 - 08:30 AM

Hi...I am new to EMSA technique..my problem is as follow:

I use a DIG Gel Shift Kit, 2nd generation; For nonradioactive detection of sequence-specific DNA binding proteins from Roche Applied Science...However...Nothing appears on my gel "niether the free labeled SSDNA "band size =90 bp" nor the Protein+ ssDNA"...only band trapeed at the well and very faint bands at the size wanted...The labeled free DsDNA as a control from the kit appears very well "band size = 39 bp".

Concentration used in my experiment as follow:

- as described in this kit: we mix control DsDNA about 1 ng with 75 ng protein pure.
while in my experiment: I mix 20 ng of SSDNA with 400 ng his tag pure recombinant protein.

I use 6% native(38"acrylamide":2"bisacrylamide")polyacrylamide gel.

I wonder If I should use a lower Polyacrylamide gel percentage...
Note: If any one has the protocol and the concentrations used for preparing native polyacrylamide gel for EMSA technique ...this will be of great help.

-I use dry bloting ...

Thanks in advance

Edited by rgn, 30 January 2011 - 08:33 AM.





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