Membranes that have been used in noncolorimetric immunodetection methods such as chemiluminescent or fluorescent techniques can be stripped of antibodies for use in subsequent rounds of Western blot detection. This allows the same blot to be probed for different proteins and saves both time and sample material. This section provides some tips for stripping and reprobing membranes used in Western blotting.
Related Topics: Antibody Selection and Dilution, Detection Methods, Western Blotting.
Blots can be stripped and reprobed several times but each round of stripping removes some sample from the blot. This decreases the sensitivity of subsequent rounds of detection and may necessitate longer exposure times or more sensitive detection methods.
When reprobing a blot multiple times:
Stripping and reprobing PVDF membranes. E. coli lysate containing human transferrin and a GST-tagged protein was loaded on a gel and blotted onto PVDF membranes. (A), the blot probed with an anti-GST antibody and developed with Immun-Star™ WesternC™ chemiluminescent substrate. (B), the same blot subsequently stripped of antibody and reprobed with the secondary antibody and developed with chemiluminescent substrate to demonstrate removal of primary antibody. This blot was also reprobed with StrepTactin-HRP to visualize the ladder. (C), the stripped blot reprobed with an anti-human transferrin antibody. (D), a control blot that did not undergo the stripping procedure probed with the anti-human transferrin antibody.
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