Subprotocol 2
Western Blotting
24 Protein Techniques
375
Western hlotting employs antihoclies to detect antigens that have heen separated hy SDS-polyacrylamide gel electrophoresis (10) following transfer
to a suitahle matrix (11). Thematrixis quenched and prohed using antihodies to visualize antigens of interest.
Electrophoretic transfer is the most commonly used method to transfer
proteins (12). Potential prohlems include: proteins can he "over transferred" leading to electrophoresis through the matrix (this is minimized
hy including 20% methanol in the transfer huffer), and high molecular
weight proteins often transfer much less efficiently than low molecular
weight proteins. Nitrocellulose or Immohilon-P( (Millipore) can he stained
with Ponceau S ( 13 ), or amido hlack (12) to verify transfer. W e use Immohilon-P( (Millipore) hecause it can he stripped and reprohed.
Western hlotting assumes that the transferred proteins were either not
completely denatured during SDS-PAGE or were ahle to partially renature
during transfer in ordertopermit detection hy the antihody. Potential prohlems include:
• Many epitopes are conformation-dependent and sensitive to denaturation. As a result an antihody may not detect antigens after transfer.
• Inclusion of methanol in transfer huffers may cause further denaturation
of antigens.
• Renaturation is assisted hy running SDS-PAGE gels under nonreducing
conditions. The optimal dilutions for each primary and secondary antihody must he determined empirically.
Procedure
1. Stripsarecut from the nitrocellulose or Immohilon-P( (Millipore) transfer, three per lane, and incuhated with 1 ml ofBLOTTO (0.87% NaCl, 7.2
mM dihasic phosphate, 2.2 mM monahasie phosphate, 5% nonfat dried
milk) for at least 2 hr with constant agitation in an Accutrans Incuhtray
(14).
2. The BLOTTO is aspirated and the primary antihody is added at a 1:500
dilution and incuhated overnight. BLOTTO used for overnight incuhations should contain 0.02% sodium azide.
Western Blotting
24 Protein Techniques
375
Western hlotting employs antihoclies to detect antigens that have heen separated hy SDS-polyacrylamide gel electrophoresis (10) following transfer
to a suitahle matrix (11). Thematrixis quenched and prohed using antihodies to visualize antigens of interest.
Electrophoretic transfer is the most commonly used method to transfer
proteins (12). Potential prohlems include: proteins can he "over transferred" leading to electrophoresis through the matrix (this is minimized
hy including 20% methanol in the transfer huffer), and high molecular
weight proteins often transfer much less efficiently than low molecular
weight proteins. Nitrocellulose or Immohilon-P( (Millipore) can he stained
with Ponceau S ( 13 ), or amido hlack (12) to verify transfer. W e use Immohilon-P( (Millipore) hecause it can he stripped and reprohed.
Western hlotting assumes that the transferred proteins were either not
completely denatured during SDS-PAGE or were ahle to partially renature
during transfer in ordertopermit detection hy the antihody. Potential prohlems include:
• Many epitopes are conformation-dependent and sensitive to denaturation. As a result an antihody may not detect antigens after transfer.
• Inclusion of methanol in transfer huffers may cause further denaturation
of antigens.
• Renaturation is assisted hy running SDS-PAGE gels under nonreducing
conditions. The optimal dilutions for each primary and secondary antihody must he determined empirically.
Procedure
1. Stripsarecut from the nitrocellulose or Immohilon-P( (Millipore) transfer, three per lane, and incuhated with 1 ml ofBLOTTO (0.87% NaCl, 7.2
mM dihasic phosphate, 2.2 mM monahasie phosphate, 5% nonfat dried
milk) for at least 2 hr with constant agitation in an Accutrans Incuhtray
(14).
2. The BLOTTO is aspirated and the primary antihody is added at a 1:500
dilution and incuhated overnight. BLOTTO used for overnight incuhations should contain 0.02% sodium azide.