2D Gel Western Blotting
Western blotting enables researchers to look at a single
protein or subset of proteins on complex 2D gels with very high
sensitivity via antibody binding. It has many uses. For example WB may
be used to follow protein phosphoryation if an antibody against a
protein binds to all the phosphorylated forms; phosphorylation causes
the protein to shift towards the acid end of the 2D gel creating
'beads-on-a-string" look for multiple phosphates. Western blotting is
also useful for
identifying known and unknown proteins in complexes brought down by co-immunoprecipitation. Once
proteins of interest are located on a Western blot, corresponding spots can
be cut from a duplicate Coomassie blue stained gel and identified by mass
packages using in house antibodies are now available:
PhosphoTyrosine Western Blotting Otherwise the client must
supply the antibody.
After 2D gel electrophoresis,
proteins are transblotted from the acrylamide slab gel to the surface of a membrane
called polyvinylidene difluoride (PVDF). The blot is stained with Coomassie
blue to show the pattern, scanned to record the image, "blocked" with a
nonreactive protein such as casein to cover nonspecific binding sites,
and finally incubated with an antibody against the protein of interest.
After excess antibody is washed away, the blot is incubated with a
secondary antibody against the primary one. The secondary ab contains
bound HRP (horse radish peroxidase) which reacts in turn with a
fluorescing reagent, ECL. The light emitted by the ECL reveals the
position of the protein of interest on an x-ray film which is
superimposable with the Coomassie blue image showing the pattern.
Our Lab Manager,
Jon Johansen, and Biochemist, Matt Hoelter, have a great deal of
experience Western blotting with a variety of proteins and antibodies.
While the success of this method depends mostly on antibody specificity,
Jon and Matt are skilled at optimizing conditions to give a good blot image with low film
background. They are
especially expert at finding corresponding proteins on duplicate Coomassie
gels for cutout and identification by mass spectrometry.
BP-6: WESTERN (IMMUNO-) BLOTTING, CLIENT SUPPLIES
ANTIBODY. This method for immuno-detection
of specific bands or spots is carried out on PVDF transblots from 1-D or 2-D
gels (see: "Handbook of Immunoblotting of Proteins Vol 1 and 11", Eds.
Bjerrum, O. and Heegaar, N., 1988, CRC Press, Inc; and "Gel Electrophoresis:
Proteins" by Dunn, M.J., 1993, Bios Scientific Publishers, Ltd). Requires
prior electrophoresis (2D-ES-1 or 1D-ES-1) but PVDF transblotting (BP-1) and
staining (BP-2) are
included in the fee. Generally the client supplies the antibody. The
sensitive ECL method of detection is used.
Standard format gel Add $290/sample.
Large Format gel, Add $370/sample.
BP-1: TRANSBLOTTING ONTO PVDF AND RETURN OF THE BLOT.
Transblotting onto PVDF is carried out
overnight according to standard procedures using a tris/glycine/methanol
buffer system without SDS. The dried stained (see BP-2) or unstained blot is
returned to the client. Staining facilitates matching to a duplicate
Coomassie gel for mass spectrometry. Original gels are stained to make sure
they’re blank and discarded, unless otherwise requested. We will change the
blotting procedure to your specifications if necessary.
Price: BP-1 Standard format, add $75 per blot; BP-1LF
Large format, add $85 per blot.
BP-2: PVDF COOMASSIE STAINING WITH ELECTRONIC PHOTOS
The 2D gel pattern obtained from Coomassie blue staining of a
PVDF membrane is faded but the scanned image can be contrasted with Adobe photoshop. The image is exactly
superimposable with the ECL film from Western blotting and helps to match a
few spots on the film to a busy 2D gel pattern on a duplicate Coomassie gel
run for mass spectrometry. Since Western blotting can be 100 times more
sensitive than Coomassie, careful matching is important. Coomassie staining
of the blot does not interfere with subsequent Western blotting.
Price: BP-2 add $13/blot. 2D-ES-14 Electronic
photos, add $16/blot. (Both, add $29/blot)
BP-3: TRANSBLOTTING ONTO NITROCELLULOSE.
Transblotting is carried out overnight according to standard
gel procedures using a tris/glycine/methanol buffer system containing 0.1%
SDS, with nitrocellulose of 0.45 micron pore size. Dried unstained blots are
returned to the client and original gels are discarded unless additional
treatments are requested. We will change the blotting procedure to your
specifications if necessary. Nitrocellulose cannot be Coomassie
Price: BP-3 Standard format gel $75 per blot; BP-3LF
Large format gel $85 per blot.
BP-5: COLORED MOLECULAR WEIGHT MARKERS FOR BLOTS.
These markers from Diversified Biotech are added during slab gel
electrophoresis; so that colored bands are visible on both unstained gels
and subsequent transblots at molecular weights of 95,500, 55,000, 43,000,
36,000, 29,000, 18,400, and 12,400.
Price: $13 per gel.