Anti-HA-Peroxidase Troubleshooting

Product No. 11667475001

Troubleshooting

No visible signal or weak signal is observed

  • Use PVDF membranes for transfer. Nitrocellulose can be used, but generally produces reduced signals.
  • Verify efficiency of protein transfer from gel to membrane by silver staining the remaining gel. To improve transfer efficiency, increase the current and/or the transfer time. Ensure there are no air bubbles between the membrane and gel during transfer.
  • Prolong the incubation time of the Anti-[HA]-Peroxidase conjugate with the membrane blot.
  • Prolong X-ray film exposure time. Luminescence reaction is almost constant for several hours.
  • Increase the concentration of the Anti-[HA]-Peroxidase conjugate by 2-fold increments.
  • Shorten the washing time; use washing buffer without Tween 20.
  • Increase the amount of protein applied to the gel.
  • Anti-[HA]-Peroxidase has been thoroughly tested for stability when stored frozen at -15 to -25 °C. However, this reagent may be inactivated by buffers containing such preservatives as sodium azide. Check peroxidase activity of the Anti-[HA]-Peroxidase conjugate. Dot different dilutions of conjugate onto a blotting membrane, and detect directly. If no signal appears, use fresh Anti-[HA]-Peroxidase conjugate, and assay in the same way.
  • It is possible the HA-tag sequence may not be accessible to detection by Anti-[HA]-Peroxidase conjugate due to conformational constraints related to the tag 's insertion site within the target protein. Verify the HA-tag sequence is correctly inserted within the target gene 's open reading frame. Consider alternative insertion sites within the target gene for the HA-tag sequence.

High background, additional bands on blot

  • Crossreacting bands have been reported in certain western-blot experiments performed with Anti-HA (12CA5). In order to determine the specificity of the Anti-HA-Peroxidase conjugate, include a negative control cell extract prepared from the host organism and lacking the HA-tagged protein being analyzed.
  • Decrease the concentration of Anti-HA-Peroxidase conjugate by twofold increments.
  • Increase washing times.
  • Shorten X-ray film exposure times.
  • Use PVDF membranes for transfer. Avoid using nylon membranes, which may lead to high background.
  • Use Carnation nonfat dry milk (5% w/v) dissolved in PBS as blocking reagent and as conjugate diluent. However, 5% nonfat dry milk may reduce specific signal as well as background.
  • Use clean equipment, freshly prepared buffers, and new membranes.

 

Materials

     
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