SDS-PAGE and Electroblotting
We currently use a Mini-PROTEAN II Dual Slab Cell system and a
Mini Trans-Blot Electrophoretic Transfer Cell system, both from
Bio-Rad Laboratories.
The following is a general description of SDS-PAGE and blotting for
amino acid analysis and N-terminal protein sequencing.
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
in the presence of a reducing agent (2-mercaptoethanol) is a
technique for the separation of polypeptide subunits according to
their molecular weight. The protocol involves denaturing the protein
sample by heating it in the presence of SDS and a reducing agent. SDS
will bind to the protein causing it to unfold, whereas the reducing
agent will reduce the intramolecular and intermolecular disulfide
bonds. The binding of SDS by the protein confers a net negative
charge and the denatured polypeptide will migrate through a gel of
known percent acrylamide in the presence of an applied electric field
towards the positive electrode (anode). After the electrophoresis is
complete, the gel is stained with Coomassie" Blue R-250 to visualize
the polypeptide bands. The molecular weight of the polypeptide is
inversely proportional to its mobility. The molecular weight of the
polypeptide subunit can be estimated directly from a semilog graph of
the molecular weight of standard proteins versus their mobility or
from a plot of the log of molecular weight versus mobility.
Separation of proteins by SDS-PAGE is an excellent technique for
producing individually "purified" proteins.
Electroblotting
Blotting is a technique for the electrophoretic transfer of DNA, RNA
or protein to a suitable membrane. The method most commonly used for
the electrotransfer of proteins to nitrocellulose is that reported by
Towbin et al. (1979). In order to take advantage of this technique
for the purpose of amino acid analysis or N-terminal sequencing, the
proteins must be transferred to a membrane that is stable to the
chemicals used in these analytical procedures. For protein sequencing
and amino acid analysis the proteins are transferred to a chemically
stable membrane, polyvinylidene difluoride (PVDF). In our laboratory
we use the "wet" transfer technique, rather than the "dry" transfer
technique. Proteins are first separated by SDS-PAGE, the gel is
removed from the electrophoresis cassette (do not stain the gel
before blotting) and equilibrated in transfer buffer without
methanol. The PVDF membrane is "activated" by dipping it in methanol;
it is then placed in transfer buffer containing methanol. The
gel-PVDF sandwich is placed in a specially designed holder that in
turn is placed in the buffer-containing electrophoresis unit. At the
pH of the buffer (pH 8.3) most proteins are negatively charged and
will migrate to the anode (positive electrode). In case one suspects
the protein has a pI greater than 8.3, a PVDF membrane can be placed
at the cathode-side of the gel as well. Alternatively, the pH of the
transfer buffer can be adjusted to a higher pH. After transfer, the
membrane is stained with Coomassie Blue R-250 and destained to locate
the protein bands. Sections containing the proteins bands can then be
excised for amino acid analysis and N-terminal protein
sequencing.
- SDS-PAGE "Hall of Shame" from Rice University-examples of gels that did not run correctly and the cause
- Smeared gels from Rice University-examples of smeared gels and the reasons
- SDS-PAGE/Blotting Submission Form (PDF)
