Protein Protocols Protein Protocols

1026 Hammerl et al.
down, with the protein bands crossing the bands on the second blot. This assembly is
wrapped in a dialysis membrane.
3. Antibodies are electrophoretically transferred from the donor blot to the receptor blot in
the presence of NaSCN, which dissociates antigen–antibody complexes.
4. The donor blot is then discarded. The receptor blot, still wrapped in the dialysis membrane,
is equilibrated with phosphate-buffered saline (PBS) to allow binding of the transferred
antibodies to the protein bands.
5. Bound antibodies are detected by use of an enzyme-linked second antibody.
This method may be useful in a wide variety of serological studies. For example, it may
help to identify structurally related molecules of different molecular weight within protein
mixtures, for example, cellular extracts. It may provide information about the serological
relationship of different antigen mixtures, and thereby help to investigate evolutionary distances.
It may also find application in analyses of subunit composition of proteins and, in
combination with peptide mapping, it can be a useful tool for epitope characterization.
2. Materials
2.1. SDS-PAGE
All solutions for SDS-PAGE should be prepared with chemicals of the highest
purity available using double-distilled water.
1. Solution A (acrylamide): 29% (w/v) acrylamide, 1% (w/v) bis-acrylamide; store at 4°C,
light sensitive, stable for approx 4 wk.
2. Solution B (separating gel buffer): 1.5 M Tris-HCl, pH 8.8; store at 4°C.
3. Solution C (stacking gel buffer): 0.5 M Tris-HCl, pH 6.8; store at 4°C.
4. SDS: 10% (w/v); store at room temperature.
5. Sample buffer: 62.5 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% SDS, 1% (v/v) of a
saturated aqueous solution of Bromophenol blue. Stored aliquots of 1 mL at –20°C may
be thawed and frozen repeatedly.
6. Electrophoresis buffer: 25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS. Do not adjust pH.
7. Dithiothreitol (DTT): 1 M Dithiotreitol. Stored aliquots of 1 mL at –20°C may be thawed
and frozen repeatedly.
8. Iodoacetamide (IAA): 1 M IAA. Stored aliquots of 1 mL at –20°C may be thawed and
frozen repeatedly.
9. N,N,N',N'-Tetramethylethylendiamine (TEMED): store at 4°C.
10. Ammonium persulfate (APS): 10% (w/v) APS. Store aliquots of 0.1 mL at –20°C.
11. n-Butanol (water saturated): Mix equal volumes of n-butanol and water and shake well.
After separation of the two phases, the upper one is water-saturated butanol. Store at room
temperature.
2.2. Electrotransfer
All buffers for this protocol contain methanol. Caution: methanol is toxic and volatile,
and should be handled in a fume hood. Wear gloves and protective clothing and do
not leave bottles open at the workbench.
1. Buffer A: 0.3 M Tris, 20% (v/v) methanol. Do not adjust pH. Store at 4°C.
2. Buffer B: 25 mM Tris, 20 % (v/v) methanol. Do not adjust pH. Store at 4°C.
3. Buffer C: 40 mM Aminocaproic acid, 25 mM Tris, 20% (v/v) methanol. Do not adjust pH.
Store at 4°C.