NFK beta primers - (Jan/10/2011 )

Dear colleagues,


Please help me. I am studying NFK beta gene expression in lymphocytes and I need quantify the mRNA expression and the protein that binding to DNA in the nucleus. I was reading about this and people usually use the Real Time PCR and EMSA techniques to analyse both mRNA and protein, respectively. Anyone could tell me primers sequence for NF Kb Real Time PCR and anyone knows some protocols to substitute the EMSA, or if is possible use the _Real Time PCR for analyse the protein binding to the DNA?
Tank you very much.

I don't know what your level is, postdoc, student, etc. for PCR primer design, I guess you should learn how to do that yourself or at least look into the literature to find whether others have designed primers for your gene.

Regarding alternative to EMSA, you can do ChIP which is very popular nowadays for studying protein-DNA interactions.

pcrman on Wed Jan 12 03:42:33 2011 said:

I've tried, but I'm finding really difficult because I can not find the sequence of the cDNA online. I know I have only complementary to the mRNA, but sometimes happens that a primer is not going well. That´s why, I am would like use something that has already been tried before. With regard to the technique to replace EMSA, i will get some informations and I will try it.
Thanks again.

Hi alzcosmar,

I am doing research on NFkB molecule.
I agree with you that it will be difficult to understand the techniques and procedure. It is even more harder to find the required information.
I am also a beginner in this field, but let me help you.

What do you have ? mRNA or cDNA ??

It is always better to start PCR first rather than going to other techniques.
If you have mRNA then synthesize cDNA first.

If you want to see the DNA sequence of each subunit lets say, p50. Go to pubmed and select nucleotide on search box. Then type p50 and select species and scroll down to see the required sequence.

If you want to design primer for each subunit of NFkB , it is rather difficult.
Some company offers to design primers for you with some extra charge.
Try looking for some references/literature and you might be lucky.

You can also go to SAbiosciences website and look for NFkB pcr array and look for various gene ID .
Hope this might be useful
Let me know if you have any doubts.
Milan

Not sure what the money situation is in your lab. ABI has taqman primer probes for the subunits of NFkB. (its not 'beta', by the way )


Another thing you could do to look at nuclear localization of your subunits of interest is to separate your protein into cytoplamic and nuclear fractions and then run western blots to see if you have nuclear shuttling.

EMSA is still required to see if those proteins that do localize to the nucleus are indeed able to bind to DNA. Not sure of a protocol substitute for this, but I haven't really looked into anything else. Why can't you run EMSA?