EMSA problems! - (Jun/21/2006 )

Hi,
I'm trying to test NF-kB activation using EMSA, and had a problem: my target band is smeared and not clear. I wonder how can I increase the signal with relatively good quality.
Here is my experimental conditions:
- 1.2ug protein from nuclear extract
- 6% native gel (0.5X TBE, 0.025% glycerol)
- Binding buffer : 20% glycerol, 5mM MgCl2, 2.5mM EDTA, 2.5mM DTT, 250mM NaCl, 50mM Tris 7.5, 0.25mg/ml poly(dI-dC).(poly(dI-dC) (followed Promega's mannual)
-Running gel: 600V, 40 minutes at room temperature.
Thank you so much for your help!

Hi,
I'm trying to test NF-kB activation using EMSA, and had a problem: my target band is smeared and not clear. I wonder how can I increase the signal with relatively good quality.
Here is my experimental conditions:
- 1.2ug protein from nuclear extract
- 6% native gel (0.5X TBE, 0.025% glycerol)
- Binding buffer : 20% glycerol, 5mM MgCl2, 2.5mM EDTA, 2.5mM DTT, 250mM NaCl, 50mM Tris 7.5, 0.25mg/ml poly(dI-dC).(poly(dI-dC) (followed Promega's mannual)
-Running gel: 600V, 40 minutes at room temperature.
Thank you so much for your help!

My protocol is like this:
- Incubate my sample with binding Buffer at Room temperature for 10min
- Add proble (label NF-kB consensus sequence) and incubate 20min more at RT
- Loading and run the gel

I wonder if I should incubate on ice or at RT? which is better?
When I ran the gel at 300V, the current is 8.6mA, hene I increased the voltage to 600V and at this point, the current was 18.6mA and it took 45 min to complete running the gel.

According to Promega's protocol, running the gel in 20 min at RT is recommended. But I am not sure if the current in my situation is too low, and if this fact has any effect on the binding?

Thanks so much,

I did EMSA, with the NF-kB probe from Promega, but not all protocol was from Promega. It was successful.

the binding buffer was 10 mM HEPES pH7.9, KCl 50 mMEDTA 0.2 mM, DTT 2.5 mM, glycerol 10%, NP40 0.05%
Incubation at RT (25°C) for 30 minutes.

Then I did the electrophoresis on a non denaturing 7% polyacrylamide gel (mini-gel)
acylamide/bisacrylamide 40% 3.5mL
TBE 10x 1 mL
glycerol 80% 624 uL
water 14.7 mL
APS 10 % 150 uL
temed 10 uL

I migrate at 150V for 1h30, at 4°C

My protocol is like this:
- Incubate my sample with binding Buffer at Room temperature for 10min
- Add proble (label NF-kB consensus sequence) and incubate 20min more at RT
- Loading and run the gel

I wonder if I should incubate on ice or at RT? which is better?
When I ran the gel at 300V, the current is 8.6mA, hene I increased the voltage to 600V and at this point, the current was 18.6mA and it took 45 min to complete running the gel.

According to Promega's protocol, running the gel in 20 min at RT is recommended. But I am not sure if the current in my situation is too low, and if this fact has any effect on the binding?

Thanks so much,

Hello,

I am beginning EMSAs with NFkB, and I was wondering if you've finally been successfull in this experiment?
After have been performing EMSAs with purified NFkB (p50), I now want to do supershift on nuclear extracts. And for the moment I did not find any good results.
I'm using 10microg of nuclear extracts and migrate my samples on the same gel as yours. And in fact I found this protocol on the web wich I'm working with http://www.celldeath.de/apometh/emsa.html... I don't know what you think of it?

So if you have some good advices for me it would be very nice!!!

Thank you very much

Flavie

Hi,
I'm trying to test NF-kB activation using EMSA, and had a problem: my target band is smeared and not clear. I wonder how can I increase the signal with relatively good quality.
Here is my experimental conditions:
- 1.2ug protein from nuclear extract
- 6% native gel (0.5X TBE, 0.025% glycerol)
- Binding buffer : 20% glycerol, 5mM MgCl2, 2.5mM EDTA, 2.5mM DTT, 250mM NaCl, 50mM Tris 7.5, 0.25mg/ml poly(dI-dC).(poly(dI-dC) (followed Promega's mannual)
-Running gel: 600V, 40 minutes at room temperature.
Thank you so much for your help!