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Anti-HA-Peroxidase, High Affinity Troubleshooting

Product No. 12013819001

Troubleshooting

Ghost Bands
Using Anti-HA-Peroxidase, High Affinity as primary antibody in an indirect detection procedure often causes so-called "ghost bands". These bands are probably the result of nonspecific binding of either the primary or the secondary antibody conjugate to other proteins in the sample (Field et al., 1988 Mol.Cell.Biol. 8: 2152-2165).
Switching to a direct detection procedure with Anti-HA-Peroxidase, High Affinity as the detecting antibody should eliminate ghost bands caused by the secondary antibody conjugate, since it eliminates the need for a secondary antibody.

Roche recommends including a negative control cell extract, prepared from the host organism but lacking the HA-tagged protein, for the western blot. With this control, albeit should be possible to distinguish a true positive signal from a nonspecific signal.

NONSPECIFIC REACTIVITY ESPECIALLY WITH HIGH TOTAL PROTEIN LOADING

  • Inadequate buffer conditions

Use PBS containing Blocking Reagent for membrane blocking and dilution of the Anti-HA-Peroxidase, High Affinity. Prolong time for blocking the membrane.

  • Inadequate washing

Increase washing time.

STAINING OF THE PROTEIN OF INTEREST IS TOO WEAK OR TOO STRONG

  • Inadequate amounts of protein loaded onto the gel; inadequate conditions used for detection
  1. Increase or decrease the amount of total protein loading
  2. Increase or decrease the concentration of Anti-HA-Peroxidase, High Affinity.
  3. Shorten or prolong exposure time used during detection.

Materials

     
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