RNA EMSA Labeling Method with Biotin

[admin]

I had a question:

i'm doing in EMSA RNA....
i'm very2 new in this field.....

however...may i know which one is the best for labelling EMSA pierce kit...

is it biotin 3' end labelling kit....
or Chemiluminescent Nucleic acid detection module...?

[admin]

My answer is since the Pierce EMSA Chemiluminescent Kit uses streptavidin to detect the EMSA, you need to use biotin labelling.

I would use either the biotin end labelling or what I actually used myself is a biotin-16-UTP label.

The end labeled method is better since you do not interfere with protein binding as much to the RNA.

The biotin-16-UTP label is quite good also however as there is a 16 linker before you get the biotin, so you don't interfere much with RNA-protein binding.

Cheers

[eyesyah84]

besides using biotin-16-UTP....
can i use biotin-11-UTP from Biotin 3' End DNA Labeling kit...fom pierce...
for RNA-EMSA.....

please help me....

[admin]

Yes biotin-11-utp is almost as good. The 16/11 is a chemical spacer to try and allow binding to the ribonucleotides as the biotin is a large chemical group, as is known to inhibit binding of RNA protein molecules to the residues/binding sites on RNA.

11 would work just as fine as 16 spacers.

[tony.brunetti]

I have a question:
I'm doing EMSA RNA by using 2 very similar biotinylated RNA probes obtained with the Pierce EMSA Chemiluminescent Kit. Both probes are very similar in sequence and size. One is 160 bases long, the other one is 200 bases long. When I measure the amount of each by OD, I obtain a very similar value, indicating that the RNA amount in each condition is approx. the same. Based on this information, when I check them on native agarose gel using ethidium bromide, the amounts are completely different. The intensity of each band at UV light is 1 for the smaller (160) probe and 5 for the bigger (200) probe. Does anyone can help me to solve this problem? Since I need to compare the activity of both probes simultaneously, in the same gel, it is important for me to have the same amount for each probe…….
Thanks a lot

[rosa9934]

Originally Posted by tony.brunetti

I have a question:
I'm doing EMSA RNA by using 2 very similar biotinylated RNA probes obtained with the Pierce EMSA Chemiluminescent Kit. Both probes are very similar in sequence and size. One is 160 bases long, the other one is 200 bases long. When I measure the amount of each by OD, I obtain a very similar value, indicating that the RNA amount in each condition is approx. the same. Based on this information, when I check them on native agarose gel using ethidium bromide, the amounts are completely different. The intensity of each band at UV light is 1 for the smaller (160) probe and 5 for the bigger (200) probe. Does anyone can help me to solve this problem? Since I need to compare the activity of both probes simultaneously, in the same gel, it is important for me to have the same amount for each probe…….
Thanks a lot

Ethidium bromide might not be an ideal method to quantify your probes. Probe dot blotting should be better to compare concentrations and labeling efficiency.