User Manual

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Isoelectric Focusing
(IEF)
7
7.1 Introduction
Isoelectric focusing (IEF) separates proteins by their net charge rather than molecular weight. Criterion
™
IEF gels are cast with Bio-Rad’s Bio-Lyte
®
ampholytes, amphoteric molecules that generate a pH
gradient across the gels. Proteins migrate to their isoelectric point (pI), the pH at which the protein
has no net charge. Criterion IEF gels contain no denaturing agents, so IEF is performed under native
conditions.
7.2 Criterion IEF Gels
7.2.1 Gel Composition
Gel buffer 2% ampholyte, pH 3–10 or 5–8
Crosslinker 3.0% C
Stacking gel None
Storage buffer diH
2
O
Shelf life ~24 weeks at 2–8°C; expiration date is printed on each cassette
7.2.2 Gel Selection Guide
IEF gel pH Range
5–8 5–8.0
3–10 4–8.5
7.3 IEF Buffers
See Appendix B for buffer formulations. Do not adjust pH unless instructed to do so.
Running buffers:
IEF cathode buffer (1x) 20 mM lysine (free base), 20 mM arginine (free base)
Dilute 100 ml 10x stock (catalog #161-0762) with 900 ml diH
2
O
IEF anode buffer (1x) 7 mM phosphoric acid
Dilute 100 ml 10x stock (catalog #161-0761) with 900 ml diH
2
O
Sample buffer 50% glycerol