how to avoid cytosolic proteins in nucleus - (May/24/2007 )
hello
I am trying to determine if a protein is only located in the nucleus. I am using western blots, but I think that maybe my nuclear preparation is contaminated with cytosolic proteins, because I get faint bands. I am lysing HeLa cells with passive lysis buffer and then centrifuge to pelett the nuclei. is there anyway to avoid contamination or determine wether my nuclei contain cytosolic proteins as well.
thanks for your help
-Squirrel-
can't you use some proteases before nucleus lysing!!
-strawberry-
QUOTE (Squirrel @ May 24 2007, 11:12 AM)
hello
I am trying to determine if a protein is only located in the nucleus. I am using western blots, but I think that maybe my nuclear preparation is contaminated with cytosolic proteins, because I get faint bands. I am lysing HeLa cells with passive lysis buffer and then centrifuge to pelett the nuclei. is there anyway to avoid contamination or determine wether my nuclei contain cytosolic proteins as well.
thanks for your help
I am trying to determine if a protein is only located in the nucleus. I am using western blots, but I think that maybe my nuclear preparation is contaminated with cytosolic proteins, because I get faint bands. I am lysing HeLa cells with passive lysis buffer and then centrifuge to pelett the nuclei. is there anyway to avoid contamination or determine wether my nuclei contain cytosolic proteins as well.
thanks for your help
isolation of organelles is more an enrichment than a purification; if your lysis step to do not harm nuclei so that their are intact, try to wash them extensivly after pelleting; you may reduce contamination from cytosol or other organelles but may not avoid any contamination...
-The Bearer-
i use now a protocol with lysis buffer and NP40.
Before resuspending pellet for nuclear proteins, i do a second wash with cytoplasmic buffer (S100) without NP40.
Then i go for nuclear proteins.
-fred_33-