I have tried homogenization, followed by centrifugation at high speeds for 40 minutes, followed by centrifugation through a concentrator filter. I suspect my BCA assay was not accurate as I had issues with lipid contamination in my sample that led to down stream problems with my western blot (namely that the loading was not equal with our GAPDH). Thanks!
my colleague at the University of Virginia, Susanna Keller works with adipose tissue. Check out her papers on how she processed tissue. good luck
Am J Physiol Endocrinol Metab. 2012 Nov 15;303(10):E1273-86. doi: 10.1152/ajpendo.00316.2012. Epub 2012 Sep 25.
Deletion of Rab GAP AS160 modifies glucose uptake and GLUT4 translocation in primary skeletal muscles and adipocytes and impairs glucose homeostasis.
Cell Metab. 2012 Dec 5;16(6):814-24. doi: 10.1016/j.cmet.2012.11.005.
Tyk2 and Stat3 regulate brown adipose tissue differentiation and obesity.
Thanks, Brandon! I will read up on them now and contact her directly if I am not clear on her methods.
no problem at all. Below is a link to her profile at UVA. I am not sure that she is on researchgate. Good luck!
http://www.medicine.virginia.edu/clinical/departments/medicine/divisions/endocrine/staff/bios/keller
There are techniques that have been published using trizol isolated proteins for western blot. I have listed 2 below. Good luck!
Biotechniques. 2007 Apr;42(4):467-70, 472.
Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage.
Efficient recovery of proteins from multiple source samples after trizol ® or trizol ® LS RNA extraction and long-term storage
André ES Simões1, Diane M Pereira1, Joana D Amaral1, Ana F Nunes1, Sofia E Gomes1, Pedro M Rodrigues1, Adrian C Lo2, Rudi D'Hooge2, Clifford J Steer3, Stephen N Thibodeau4, Pedro M Borralho15* and Cecília MP Rodrigues15
*
Thanks for the link to her page, Brandon...and the added info on the trizol samples. Appreciate it!
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