Hi all,
I would like to extract total protein from insect faecal pellet and quantify using Bradford method. I have found a paper that extract protein using 0.1mol NaOH, but NaOH will interfere with the Bio-Rad protein assay kit in my lab. So I wonder if there is any other method that I can use to solubilise protein for quantification.
Thanks in advance.
I will grind the pellet before extraction. Will sonication help in extracting proteins?
Hi
Have a look to this publication:
FastPrep method
Proteins were extracted by using the FP120 FastPrep bead-beater
(BIO101/Savent) by alkaline-lysis protocol. In brief, the pellets
resuspended in 400 μl rehydration buffer including 8 M Urea, 2 M
Thiourea, 0.5% CHAPS, 10 mM DTT and 0.1% immobilized pH
gradient (IPG). Cells were lysed by vortexing them with acid
washed glass beads with a diameter of 212 to 300 μm (Biospec
Products, Inc.) by use of FastPrep FP120 device at 6 m/s for 3×45
s at 4°C, each 3 min in between which the cell suspension was kept
on ice for 1 min. The cell debris was removed by centrifugation at
14000×g for 10 min at 4°C (Larsen et al., 2006). The supernatant
containing protein extracts was stored at -20°C until further
analysis.
www.ajol.info/index.php/ajb/article/.../82540
Greatings
Natasha