Hi all, could anyone has idea what could be the reason for getting cytoplasmic contamination in nuclear fractions.
I am making fractions of Raw as well Thp-1 cells but I am getting bands with GAPDH as well with nuclear fractions as same as intense with cytoplasmic ones.
There is more or less some contamination with NE-PER kit. Each step must be performed very carefully to avoid contamination, especially of nuclear fraction. Use perfectly centrifuged pellet and follow correctly the duration of each treatment. Use Hsp90a and as a marker of cytoplasmic proteins and HDAC2 for proteins in nuclear fraction. In our hands the kit worked well with several cancer cell lines.
I can't use GAPDH as cytoplasmic ad lamin for nuclear?
Is we need to be so particular for vortex time like 5 sec, I min incubation or it may vary from 5 to 10sec etc.
you may use GAPDH (we have not tested lamin) too; but we found that Hsp90a and HDAC2 (from R&D Systems) give better results
no problems, such variations in vortexing are possible.
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