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  2. → Viewing Profile: Topics: silvepa23

silvepa23

Member Since 24 May 2011
Offline Last Active Feb 21 2013 07:39 AM
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Topics I've Started

cytoplasmic and nuclear extraction

20 February 2013 - 10:20 PM

Hi,

i have been using the NE-PER Nuclear and Cytoplasmic Extraction Reagents by thermo scientific Pierce.  When I run both the nuclear (15 ug) and cytoplasmic (40 ug) extracts I use b-actin as a control.  When I expose the membrane I get very strong signal in the nuclear extracts and weaker signal in the cytoplasmic despite the fact that i added more than 2x the cytoplasmic protein to the gel and there should be more in the cytoplasm.  has anyone else used this kit and had a similar problem.  thanks

western blot and actin issue

10 January 2013 - 08:20 PM

Hi,

I would greatly appreciate any help.  I recently infected some mammary epithelial cells and harvest the protein.  i use the quibit quantification to determine the protein concentration of all my samples.  i ran a western blot and probed for b-actin.  the b-actin were not equal.  i have had trouble in the past with the quibit and nanodrop.  i was wondering if i can just chose a volume of protein (not equal conc based on quibit quantification), run a western blot, and then use imaje j to adjust the volume?  So if i get all the density form image just can't i just standardize them to one of value and change the rest or is there not a linear relationship between protein concentration and band intensity?  thanks.

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