I have used a protocol from C. L. German and C. L. Howe (2009) with some modifications. To be brief, I didn't use an ultracentrifuge at the end with the cytosolic fraction. I stained for histone H3, HSP70 and for b-actin. I see H3 only in nuclear fraction, but HSP70 and b-actin in both cytosolic and nuclear. My cytosolic fraction can have some of the small vesicles fraction proteins, but that is not a problem in my experiment. I need a good marker that is only in cytoplasm and not in nucleus/ER. I know that my cytosolic fraction is free from nucleus contamination, as there is no H3 there, so I feel okay about that protocol, but I can't imagine a situation in which I would have almost the same amount of cytoplasmic proteins in my cytosolic and nuclear fraction. I was thinking that b-actin will work great and now I don't know if it can show up in nucleus.
I will be grateful for any help and I hope I made myself understandable.
In our hands Hsp90a and no b-actin worked well as a marker of cytoplasmic proteins and HDAC2 for nuclear fractions. Depending on the extraction method, there is more or less some contamination in the samples. Each step must be performed very carefully to avoid contamination, especially nuclear fraction. There is a kit from Thermo that works rather good to get rapidly nuclear and cytoplasmic fractions.
We use GAPDH as a cytosolic marker. A very good antibody with a strong reaction and it shows no presence in the nucleus.
Good luck!
I guess I should have report something after I've listened to your answers. In case someone is reading it later:
GAPDH worked perfect as cytosolic marker and the protocol I used resulted in no detectable cross-contamination of fractions. There was no GAPDH in nuclear fraction and no H3 in cytosolic fraction.
Dear Kajetan, could you maybe share the protocol you used to separate nuclear and cytosolic fraction?
Dear Felix, I am terribly sorry to answer that late, I haven't noticed the notification from researchgate. I started off with several papers that have shown fractionation and chose one to apply it with modifications: "Preparation of Biologically Active Subcellular Fractions Using the Balch Homogenizer" by CL German and CL Howe, 2009.
I used buffers as described there. Just after decapitating animals I harvested tissue and put it in liquid nitrogen swiftly (in Eppendorf tubes, of course), kept them in -80 before next procedures. I weighted the tissue and calculated buffer volumes: for 100 mg of tissue I have used 2 ml of MES buffer and 600 ul of both NDG and RIPA in later steps. I used glass-glass Potter-Elvehjem homogenizer, using around 20 strokes, until suspension was clear. Afterwards I centrifuged samples at as described in the paper, regarding nuclear fraction. To get cytosolic fraction instead of final step of ultracentrifugation, I just used max g in my centrifuge (~22000 g) for 30 min and I've used the supernatant as cytosolic fraction, without any precipitation. I got concentrations around 2 ug/ul for both fractions, I haven't used others (ghost, big/small vesicles).
Sorry for late reply.
I used GAPDH, Alpha tubulin, and Beta actin as cytosolic markers and Lamin A/C, and Histone H3 as nuclear markers for my cytosolic and nuclear fractions. I got intense bands for GAPDH, alpha tubulin and beta actin in my cytosolic extract. However, i saw faint bands in my nuclear extract too with those cytosolic markers. That might be because of the presence of little bit of cytosolic fraction in my nuclear extract. I was mostly satisfied with alpha tubulin and would recommend that. On the other hand, I obtained very nice, intense bands for Histone H3 and Lamin A/C for nuclear fraction without any bands in cytoplasmic fraction. I was mostly satisfied with Lamin A/C and recommend that.
Simona John von Freyend Hi Simona,
Which manufacture do you use? I would be grateful if you can provide me the reference number.
Thanks a lot in advance!
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