SDS-PAGE
(Sodium dodecyl sulfate- polyacrylamide gel electrophoresis)
Alex de Lencastre
Bio 606
9/26/2013
Learning Objectives
This week:
4. Estimation of purity and expression of our 6-His-GBP
protein using SDS-PAGE.
Next week: REVIEW
Following week: EXAM - I & Notebook Grading - I
PAGE
PAGE = Polyacrylamide Gel Electrophoresis
Under “non-denaturing”
or “native” conditions,
rate of movement in gel
depends on size, shape
and charge.
COMPLICATED!
PAGE – native or denaturing
8% 10%
“Discontinous PAGE”
Running gel
Stacking Gel: pH (6.8) Running Gel: pH (8.8)
Glycine net charge is Glycine net charge is
barely negative much more negative
Glycine ionization
Net charge
+1 0 -1
A set of 10 pre-stained protein bands, with known size, in the range of 6-180kDa
Run your gel at the same time as the protein ladder (standards) and then compare
the size of your protein to the size of the standards.
Loading a gel
Your sample +
- SDS
- BME
- Glycerol
- Dyes
Silver stain
Slow (2-3 hours)
Detection limit: 100 times greater than Coomassie
Example SDS-PAGE Gel
Predict:
What do you think gel
will look like?