Nuclear fraction versus cytoplasmic fraction - (Feb/03/2011 )

Hello,

I am trying to run a western blot of the nfkb protein p105/p50. the p105 is a precursor that is cleaved to p50 which is the active subunit that enters the nucleus. I have been trying to do nuclear extractions and I figured since I don't have any nuclear protein specific antibodies why not use this p105/p50 Ab and run a western to check for the 50kDa bands between a nuclear and cytoplasmic extract as a way to validate my extraction.

My postdoc pointed out to me that my design of having 4 lanes of 0.5, 1, 3, 5ug of protein would not be comparable between nuclear and cytoplasmic fractions because the cytoplasm has naturally more proteins. I agree but what would be the best way to make this comparable? Should I do this by percentage? Figure out the total protein and scale the cytoplasmic loading proteins up?

But i guess honestly, if I am only interested in the nuclear fraction there really is no point in checking the response of the cytoplasmic? I figured it would at least provide some sort of internal control for the antibody I am using (ie expect less in the cytoplasm versus the nucleus).

Thanks!

Hi,

What is the end desire of your experiment.
The concept of p105/p50 is very complex and they seems to shuttle between cytoplasm and nucleus even without activation.
There are some newer techniques like ELISA based detection of subunits.
Try to see NFKB elisa kit from Active Motif ( bit expensive) but it detects phosphorylated subunits ( which are activated subunits) .
I have seen articles regarding p65 phosphorylation . But detection of p50/p105 is not that simple. p50 is not an active subunit. It does not have DNA binding region.
It acts a repressor subunit.
Better idea would be to see p65/p50. Unless you are specific for your research
Milan

thanks for your response

yea you are right that the p50 is shuttled in and out regardless of being phosphorylated. I think I am merely interested in the nuclear fraction versus the cytoplasmic and thus it's not necessarily important that they are phosphorylated. I did look at that TransAM kit and their results showed some low basal levels with higher nuclear fraction of p50 and amplification with induction of TNFalpha.

so far using whole cell extracts it seems the non-cancer cells do not have any p50/p105 i can detect using only 10ug of protein. I am going to try up to 50ug. Even with stimulation of TNFalpha for 30 min did not show anything.

i did just get p65 antibody last week so i will check the localization but of course i will try whole cell extracts first.

I am very new to the field of research.
I am first year PhD student (just been 5 months) and I am working on p50 KO model. This is very new concept for me now.

My plan is to work on real time PCR and determine gene expression of individual subunit first then i will go to other specific techniques.
I am not sure about the concept of cytoplasm vs nuclear fraction. As these molecules seems to shuttle continuously even without stimulation.

So far, I could find the reference of p105 and p100 primer design.
One huge question existing in my mid is - can p50 expression/regulation persists even when p105 (its precursor) is knock out ??

Thanks a lot
Milan

congrats on PhD i wish i was in a PhD program. I am only a fourth year medical student trying to find my way in lab. I don't get much teaching so I have to read everything on my own.

i too am having a growing interest in p50/p105. From my own readings my understanding is similar to yours. p50 is present at a basal level in order to act as a repressor for itself and other downstream genes i believe. However, as I mentioned previously, the Active Motif TransAm kit shows in their technical sheet that p50 can be induced with TNFalpha.

i think you have an interesting question at hand which may or may not be addressed in the literature. I am thinking can p50 be produced from another source other than activation and cleavage of the p105 precursor?

i did want to clarify that although p50 is shuttled into the nucleus without post-translational signaling (i believe) it is being shuttled as a homodimer(p50/p50) and not as a lone subunit(p50).

Thank you .
Well I was medical student and never dreamt that I will be doing research. But i found out to be more interesting and demanding at the same time.

Yes of course various heterodimer and homodimer of NFkB subunit exist to regulate various gene expression. To my understanding, no single subunit is reported so far in literature in gene expression.
The signaling pathway is very complex with various coactivators and repressors.
Recently p50:p50:Bcl3 heterodimer was shown to be transcriptionally active in producing Bcl-2.

If you think of p105, my believe is that multiple ankyrin repeats on p105 ( C terminus) behaves like IkB. So there might be two step inhibitory effect. First mediated by p105 and later by IkB.
My question is exactly the same as yours, I too am wondering about different source of p50 (other than p105).

I am in favor of p50/p105 subunits ( its homo and heterodimers) having inhibitory effects, but some journals reported just opposite. Probably, there is other source of p50 synthesis and some unknown inhibitor forming heterodimers with NFkB.

try reading missing pice in NFkB puzzle by Alexander Hoffman. He is excellent professor in Immunology and he has plenty of research regarding this field.
If you are interested more, you should try Transcription factor NFkB , a book by Sankar Ghosh, who is professor of Immunology at Yale University.

I will more than happy to share my understanding .
Milan

thanks for the suggestions i'm always looking for good reads i think i have read Hoffman's paper many months ago but clearly I need to refresh my memory.