Anti-HA Troubleshooting

Product No. ROAHA

Troubleshooting

Chemiluminescent or chromogenic signal weak or not visible

• Poor isolation of tagged protein
  — Use a different cell lysis procedure

• Antibody too dilute
  — Double the concentration of the Anti-HA and/or the secondary antibody

• Too little protein on the gel
  — Add more protein to gel.

• Poor transfer of proteins from gel to membrane
  — Increase the electrical current and/or the transfer time for the blot. Be sure there are no air bubbles between the membrane and gel during transfer.

• Wrong type of membrane
  — For maximum signal, use PVDF membranes for transfer.

• Antibody incubation too short
  — Incubate Anti-HA and/or the secondary antibody with the membrane blot for a longer time.

• Signal development time too short
  — Double the development time.

• Wash time too long or too stringent
  — Shorten the washing time. Omit Tween 20 from the Wash Buffer.

• Enzyme on antibody conjugate inactivated by preservative
  — Do not use sodium azide in any Western blot reagent if you use Peroxidase-conjugated antibodies.

• Substrate inactive
  — Make fresh dilution of substrate or start with a different stock of substrate.

• Epitope tag sequence is not detectable due to Proteolytic cleavage
  — Include protease inhibitors in lysis buffer.

• Epitope tag sequence is not detectable due to low level of expression
  — Use alternative expression system or optimize your expression system. Insert multiple tag sequences into target protein to increase avidity of antibody reaction.

• Epitope tag sequence is not detectable due to premature translation termination resulting in loss of C-terminal tag
  — Use alternative insertion site within the target gene for the epitope tag sequence.

High background additional bands on blot

• Antibody too concentrated
  — Reduce concentration of Anti-HA and/or secondary antibody by half.

• Wash time too short
  — Prolong wash time.Incubation of membrane with substrate too long — Leave blot membrane in substrate for a shorter time.

• Wrong type of membrane
  — For minimum background, use PVDF membranes for transfer.

• Blocking Reagent too dilute
  — Use nonfat dry milk (5% w/v) dissolved in Reagent Diluent as Blocking Reagent. Note: High concentrations of nonfat dry milk may reduce specific signal as well as background.

• Contaminated reagents or equipment
  — Use clean equipment, freshly prepared buffers, and new membranes. Always avoid touching membranes with bare hands; use gloves and forceps.

• Secondary antibody binds untagged proteins.
  — Use an F(ab′)₂ fragment of a secondary antibody, rather than an intact IgG.

• Heavy and light chains of primary antibody visible on blot membrane
  — Use direct detection with peroxidase-conjugated monoclonal antibody to visualize tagged proteins.

• Signal development time too long
  — Reduce development time by half.