Problems with Extraction technique

[Mercury33]

Hi, I just want to know if anyone has ever done this nuclear and cytoplasm technique? I have repeated this almost 15 times over a course of 4 months with the same result i.e. very low result for the nuclear so not sure whether everything has gone into the cytoplasm or is it just a case of cell number. I have checked the Buffer A, Buffer A & Igepal along with Buffer C and Buffer D. If anyone has come across or done this can they let me know if they had similar problems? I'd like to get to the bottom of it & see are there any other alternatives coz it's honestly driving me mad! Many thanks

[JackBean]

which technique?

[Mercury33]

I want to get nuclear and cytoplasmic fractions - this is the technique however, Bradford readings tell me that everything is going into the cytoplasm (very high values 0.5, 0.6 etc.) and my nuclear is 0.295, 0.33 etc. This is almost the same as my BLANK which is WATER (sorry just writing capitals to emphasize). It's like everything is going into the cytoplasm (i.e. both cytoplasm and nuclear together). This could also happen with a low number of cells but I need to ask if anyone has anything to suggest as I am going mad repeating on that scale - it takes about 4 days to grow cells and this takes approx. 2-3 hrs - imagine repeating on a daily, weekly basis!! Anyone got anything to offer for new kit etc. please send a reply Many thanks

[canalon]

Yes it is clear that you are doing cell fractionation. But what protocol are you using. There are more than one.

[Mercury33]

Ok this sounds silly but my supervisor literally just gave me this protocol called nuclear extraction (it involves scraping cells off plate in PBS, spinning down - taking off supernatant & resuspending in Buffer A (contains Hepes, MgCl2, KCl2) along with PMSF and DTT, you spin again, take off supernatant and resuspend pellet in Buffer A with Igepal (very viscous to break open cells) - leave on ice, centrifuge - supernatant is the cytoplasmic fraction. Next, resuspend the pellet in Buffer C (contains PMSF), leave on ice for 15 mins then centrufuge again - supernatant is the nuclear extract - you add this to Buffer D in separate tubes already containin PMSF and DTT. You then run Bradford - results which I explained above

[MJD]

A couple of questions. How much Buffer D are you adding to the nuclear fraction? Secondly, How many cells are you growing for this protocol? The blank for your spectrophotometer shouldn't be that high- there seems to be substantial background. 10 million cells should give you sufficient protein just be careful how much buffer you are adding to the nuclear fraction. Even though the protocol at the following link using NP-40, not IGEPAL, look at the volume of Buffer C and D to get an idea if you may be diluting the nuclear fraction too much. What would be typical protein concentration for the numbers you mentioned in your post for the cytoplasmic and nuclear fractions? What is the reason why you need to do nuclear/cytoplamic fractionation? Please explain. There may be an alternative.


https://webspace.utexas.edu/mjd57/www/N ... action.pdf

Cheers.