I need to check transcriptional activation of NFkB in rat brain.
An easy way is to use nuclear isolation to separate the fractions. Alternatively, there are good kits available to separate up to 5 fractions.
The first thing you would need to do is to isolate the two hippocampus structures by dissection of the brain, which is relatively easy to do if you know how. Then, proceed with fractionation protocols/kits, as suggested by Kim van der Linde.
You can try a differential isolation by centrifugation. It´s less expensive a may be adecuate for your purpose.
Homogenate,
Centrifugation 500 x g 2 min, 4ºC. Take the supernatant
Centrifugation 500 x g, 5 min 4ºC. Save the the pellet (nuclear fraction). and take the supernatant
Centrifugation of supernatant 25.000-30.000 x g 45-60 min. Save the supernatant (cytosolic fraction).
Beside others, Cox et al (Nature Protocols, 2006; 1(4):1872-78) described a suitable method to discriminate between cytoplasmatic and nuclear protein fractions. But keep in mind, that under normal lab conditions a protein fractionation represents only an enrichment and no absolute separation of the fractions of interest!
Good luck and best regards
Frank
Centrifugal separation, and including a dense material like sucrose, should help to considerably enrich the fractions
I used a nuclear extraction NE-PER Kit (Pierce). It is very easy to use, you just need to follow the manufacturers protocol.
You can use a standard sub-cellular fraction method by sucrose gradient and differential centrifugation as suggested by Dr.Ruano. Under those protocols the first centrifugation of the homogenate can be at a lower speed to remove large debris prior to collection of the nuclear pellet. The problem is the small amount of tissue and thus, the small amount of volume. Years ago there were some methods available using an airfuge for the centrifugation. While there are a lot of "methods" sources available you may want to also check back to Methods in Enzymology which is where all of this type of information use to be published. I find it an excellent resource when I had something that I needed to work up as it is very broad based. If you can afford a commercial kit for small sample size that would probably be the best approach for overall time and money. You can work out the methods using an equal sized/weight section of the cortex until you have the method established in your hands. I would not use other brain regions as the myelin content will significantly alter the separation.
Do you have the option of looking at the structure by immunofluorescence to identify phosphorylated NFkB in the nucleus. You could consider a first step of looking at the homogenate for total versus phosphorylated NFkB - as you will likely need to use the same approach even if you have the fractions given, as mentioned earlier that the fractions are not absolute but enriched.
We have used in the past this kit:
http://www.piercenet.com/browse.cfm?fldID=06010435
It works well
Homogenise both hippocampii in 1% triton-x containing lysis buffer. Incubate on ice for 30 mins with intermittent vortex. Step1: Centrifuge the lysate at 100g for 5 mins to pellet unlysed cells. Step 2: Centrifuge the supernatant at 1500g for 15 mins to pellet nuclei. Supernatant serves as crude cytosolic fraction. Resuspend the nuclear pellet in lysis buffer again and again spin down to get the crude nuclear pellet. It is a crude method of isolation but the fractions are decently pure to work with. Best
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