western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students
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Efficient
cell membrane protein extraction is important for sample preparation and
subsequent analysis by western blot protocol. When resuspending the pellet,
use as low a volume of resuspension buffer as possible to obtain a high
protein concentration sample.
1. PubMed:
comprehensive database - 15 million citations
2. NIH:the National Institutes of Health
3. UniProt: The world's most comprehensive catalogue of information on proteins
4. Western
Blot Procedure Western blot protocol overview.
b. Use liquid nitrogen to fast freeze the sample
c. Grind the tissue with a mortar and pestle
d. Place the now powdered tissue into 10 ml lysis buffer
e. Using a polytron, homogenize the ground tissue and lysis buffer (one burst – 20 seconds)
f . Centrifuge for 15 minutes at 500 x G at 4 degrees Celsius
g. Remove and keep the supernatant
h. Homogenize the pellet again with additional 5ml lysis buffer (20 seconds)
i. Centrifuge for 15 minutes at 500 x G at 4 degrees Celsius
j. Pool both supernatants
k. Centrifuge the supernatants at 45 000 x G for 15 minutes
l. Wash the pellet twice in lysis buffer – removing the supernatant between washes
m. Resuspend in resuspension buffer
n. Store at -80oC; thaw for western blot analysis (aliquot to avoid multiple freeze-thaw cycles)