EMSA problem - (Dec/28/2007 )
I am using DIG band shift kit of Roche to test the binding of a Zinc-finger protein on a promoter. The probe is about 30bp, I use partially purified protein purified with HIS column. I added 5uM Zinc Sulfate when I purified the protein and in the binding reaction system. For the reactions, I let the reaction stay 30min on ice and then add probe for another 20min at room temperature. In the reaction system, 50nanogram of polyd(I-C) included. I use loading buffer without Bromphonel blue and run at 200V for 40 min in 4 degree with the 6% retardance gel from Invitrogen in 0.5xTBS. But unfortunately, I have not yet get see any band in my samples. In the sample added the protein, the probe had a longer tail than the probe only lane. And after added about 100ng of competing DNA, it looks like a weak band appeared. Could anybody give me some suggestion? Why do a weak band appear after adding competing DNA? Thank you for any advice.
-Daniel5306-
can you see unbinded DIg probe on your film?
QUOTE (Daniel5306 @ Dec 28 2007, 09:21 AM)
I am using DIG band shift kit of Roche to test the binding of a Zinc-finger protein on a promoter. The probe is about 30bp, I use partially purified protein purified with HIS column. I added 5uM Zinc Sulfate when I purified the protein and in the binding reaction system. For the reactions, I let the reaction stay 30min on ice and then add probe for another 20min at room temperature. In the reaction system, 50nanogram of polyd(I-C) included. I use loading buffer without Bromphonel blue and run at 200V for 40 min in 4 degree with the 6% retardance gel from Invitrogen in 0.5xTBS. But unfortunately, I have not yet get see any band in my samples. In the sample added the protein, the probe had a longer tail than the probe only lane. And after added about 100ng of competing DNA, it looks like a weak band appeared. Could anybody give me some suggestion? Why do a weak band appear after adding competing DNA? Thank you for any advice.
-cnbeatles-
Yes, I can.
QUOTE (cnbeatles @ Jan 1 2008, 10:45 AM)
can you see unbinded DIg probe on your film?
QUOTE (Daniel5306 @ Dec 28 2007, 09:21 AM)
I am using DIG band shift kit of Roche to test the binding of a Zinc-finger protein on a promoter. The probe is about 30bp, I use partially purified protein purified with HIS column. I added 5uM Zinc Sulfate when I purified the protein and in the binding reaction system. For the reactions, I let the reaction stay 30min on ice and then add probe for another 20min at room temperature. In the reaction system, 50nanogram of polyd(I-C) included. I use loading buffer without Bromphonel blue and run at 200V for 40 min in 4 degree with the 6% retardance gel from Invitrogen in 0.5xTBS. But unfortunately, I have not yet get see any band in my samples. In the sample added the protein, the probe had a longer tail than the probe only lane. And after added about 100ng of competing DNA, it looks like a weak band appeared. Could anybody give me some suggestion? Why do a weak band appear after adding competing DNA? Thank you for any advice.
-Daniel5306-
QUOTE (Daniel5306 @ Jan 2 2008, 04:27 PM)
Yes, I can.
QUOTE (cnbeatles @ Jan 1 2008, 10:45 AM)
can you see unbinded DIg probe on your film?
QUOTE (Daniel5306 @ Dec 28 2007, 09:21 AM)
I am using DIG band shift kit of Roche to test the binding of a Zinc-finger protein on a promoter. The probe is about 30bp, I use partially purified protein purified with HIS column. I added 5uM Zinc Sulfate when I purified the protein and in the binding reaction system. For the reactions, I let the reaction stay 30min on ice and then add probe for another 20min at room temperature. In the reaction system, 50nanogram of polyd(I-C) included. I use loading buffer without Bromphonel blue and run at 200V for 40 min in 4 degree with the 6% retardance gel from Invitrogen in 0.5xTBS. But unfortunately, I have not yet get see any band in my samples. In the sample added the protein, the probe had a longer tail than the probe only lane. And after added about 100ng of competing DNA, it looks like a weak band appeared. Could anybody give me some suggestion? Why do a weak band appear after adding competing DNA? Thank you for any advice.
Are your positive controls working?
-Clare-
QUOTE (Daniel5306 @ Dec 28 2007, 10:21 AM)
I am using DIG band shift kit of Roche to test the binding of a Zinc-finger protein on a promoter. The probe is about 30bp, I use partially purified protein purified with HIS column. I added 5uM Zinc Sulfate when I purified the protein and in the binding reaction system. For the reactions, I let the reaction stay 30min on ice and then add probe for another 20min at room temperature. In the reaction system, 50nanogram of polyd(I-C) included. I use loading buffer without Bromphonel blue and run at 200V for 40 min in 4 degree with the 6% retardance gel from Invitrogen in 0.5xTBS. But unfortunately, I have not yet get see any band in my samples. In the sample added the protein, the probe had a longer tail than the probe only lane. And after added about 100ng of competing DNA, it looks like a weak band appeared. Could anybody give me some suggestion? Why do a weak band appear after adding competing DNA? Thank you for any advice.
EMSA kit ofen contains a positive control.
-glcui-