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.

Materials

     
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