The EMSA image shows that you cant rely on the data as there is lot of streaking in gel. The intensities obtained from such an image will not be very reliable. Use radio labelled probe and lower amount of probe to get cleaner images. aggregates in well are also protein DNA complex.

Please try filter binding assay as alternative to determine Kd

Hi,

I can see your DNA does not form a nice band ever without the protein. It will cause troubles at every step in the downstream application.

Are you sure about the DNA concentration?

Although you didn't mention about the length of the DNA probe, 40 µM is equal to ~ 2.6 µg/µl with 100 bp dsDNA probe. Is it really that high?

How many micro-litre of such reaction mix loaded to the gel? (In other words, how much DNA did you load the gel?)

Acrylamide gels are easily saturated with large quantity of DNA. You should try to reduce the amount of DNA loaded.

You should be able to detect 100 ng of DNA easily by EtBr stain.

How many seconds did you need to obtain the image you attached?

Dineshkumar Dandekar Thank you for your suggestion. I will try to use a different technique.

Shin-Ichiro Hiraga Thank you for your comments. Regarding your valuable questions:

1- length of the DNA probe is 16pb (equal of 420 ng)

2- Indeed the quantities are really high but the binding is very weak, so when I am lowering DNA quantities I am not able to see the complex protein-DNA

3- I tried to lower the amount up to 200ng ( iwas able to detect the complex)

( I am attaching the picture of this gel)

Moreover, I tried to make the same trial with longer DNA (58ds). I was able to decrease in this case DNA quantities (70ng) (Attached as well)

When I was trying to find the best fitting of my data, I found that the specific binding with Hill Slope was much better than all to other equations.

I don't know exactly what are the needed information to decide which equation to use. Is it just a matter of fitting?

I really appreciate your help

Any comment and suggestion could be helpful and appreciated

Thank you

Joelle AYOUB

Hi,

It is good that you have clearer images using lower DNA concentration and longer probes, as it will make the band quantification easier.

As I understand, you cannot really measure Kd using EMSA, because the local concentrations of the DNA and protein change during electrophoresis, and there's now way to measure them.

For that reason, I would not take DNA concentration in consideration.

Instead, I would use hill curve to determine the "midpoint" under a particular condition. But I would not consider it as "Kd".

You would be able to use this "midpoint" value for qualitative or semi-quantitative assessment (e.g., comparing WT protein and mutant protein).

Shin-Ichiro Hiraga Thank you so much for your help and the time you gave replying to my questions. I really appreciate it.

Joelle