NF-kB protein nuclear and cytosol isolation - No band separation in cytosol and nucleus (Aug/28/2006 )
Hi,
I am recently working to see the effect of my treatment on the NF- B signaling.
First I am doing the isolation of cytoplasmic and nuclear proteins. And then I subjected to electrophoresis on 10% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane, but after transfer I have seen just single band in staining with Ponceau S in my nitrocellulosis membrane in cytosol and in nucleus. However when I did the whole cell lysate I saw in Ponceau staining separate bands. Is there any methodology to solve this trouble? Or do I am suppose just one band in cytosol and in nucleus?
Hi Kara,
I make nuclear and cytosolic fractions routinely for EMSA and western blot. I've never stained the nitrocellulose membrane with ponceau before so I don't know what to expect. I perform protein quantitation before loading equal amounts of protein then use an antibody against a loading control (PARP for nuclear, tubulin/GAPDH cytosol). How are you going to assess NFkappaB activation? p65 or p50 translocation into the nucleus or IkappaB alpha or beta degradation? I would go ahead and try the blots if you are confident of your extraction protocol.
All the best,
Ceri
I make nuclear and cytosolic fractions routinely for EMSA and western blot. I've never stained the nitrocellulose membrane with ponceau before so I don't know what to expect. I perform protein quantitation before loading equal amounts of protein then use an antibody against a loading control (PARP for nuclear, tubulin/GAPDH cytosol). How are you going to assess NFkappaB activation? p65 or p50 translocation into the nucleus or IkappaB alpha or beta degradation? I would go ahead and try the blots if you are confident of your extraction protocol.
All the best,
Ceri
Hi Ceri,
thank you for your tips. regarding you question i try to do this
Cytosols were examined for IkB , p50, and p65 NF- B, and nuclear extracts were examined for p50 and p65 NF- B . The data suppose to how that RANKL induces nuclear translocation of p50 and p65 subunits, decreases the cytoplasmic levels of these two proteins, and also degrades I kB. Consistent with my treatment (DPCPX) inhibition of phosphorylation and degradation of I kB, DPCPX when added before RANKL, totally prevents the nuclear translocation of p50 and p65 and causes these proteins to accumulate in the cytoplasm and prevent the degradation of Ik B.
i think blots is a very good idea. i would try it.
Kara
Sorry, Kara. Have you already done the blots for p65, p50 and IkBalpaha with the RANKL+/- DPCPX?
If so, I wouldn't worry about the ponceau staining I'd use another antibody against GAPDH/tubulin for the cytoplasmic extracts or PARP or laminin for the nuclear extracts to control for equal loading.
You could also use EMSAs (radioactive labelled probe or a non-radioactive biotin labelled probe of the NFkappaB consensus site). Amikins has put the non-radioactive protocols on the protocol sections of Bioforum.
All the best,
Ceri
No I have not. I just started this week. I just added today the p50 Ab . 
I did not see any band in my gel. I guess this because the protein did not separate as the ponceau stain showed. I am going to tray the EMSA. 
Honestly I don’t know how to do it. I have never done it before. is the same methodes and reagent as WB except that i should use a radioactive secundary Ab. right?I was looking for the protocol from Amikins but I could not fined it, could you please paste it again when you replaying.
Thanks Ceri
Firas
Firas,
I wouldn't give up on the blots yet. Try to optimise detecting p50 and p65 using a whole cell extract. You should be able to separate a nuclear or cytosolic extract by SDS PAGE. If you could give some more details of the protocols your using to make the extracts, how you quantitated the protein in the extracts, how much you loaded per lane, what % gel etc ? If you've just started is someone in the lab giving you methods and teaching you the techniques.
The electrophetic mobility shift assay (EMSA) or band shift uses a labelled DNA probe containing the consensus binding site for a transcription factor (either labelled with biotin or radioactive nucleotide or ATP). The probe and nuclear extract are mixed in a binding buffer and run on a native gel. The activated transcription factor complex should make a complex with the probe and run slower into the gel so it's "shifted" up away from the free probe.
Amy's protocol and some other are on the protocols section accessed from the protocol online home page in the molecular biology: DNA-protein interactions: Band shift. I can send you our protocols if you'd like also.
All the best,
Ceri
Hi Ceri,
Thanks I found the protocols that you talking about.
I made 10% gel. I load as fellow
Add 5 µl of loading buffer and 20 µl of samples to eppendorfs tubs.
Boil samples for 3-5 minutes.
Spin down for 30 second.
Run it at 65 volt for 2 hours and then I transferred in Electrophorese in the refrigerator at 100V for 1 hour. After that I checked with the Poceau stain.
I incubate with 5%NFM for 2 hours then I add Ab overnight and secondary Ab for 2 hours and I use 2 ml ECF substrate to analyse the membrane.
Thanks Ceri and have nice long weekend
Faros
Thanks Faros I had a good weekend hope you did too. Your protocol looks fine. One thing I thought of was maybe you should quantitate how much protein is in your samples (bradford or BCA) then add equal amounts of protein made up to the same volume with distilled water and loading buffer. It might may it easier to compare a whole cell extract with a cytosolic or nuclear one and also the nuclear extraction buffers have a high conc of NaCl which may effect the electrophorese possibly.
All the best,
Ceri