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.