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Anti-c-myc-Peroxidase Troubleshooting

Product No. 11814150001

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-c-myc Peroxidase.

Too little protein on the gel:
Add more protein to gel.

Poor transfer of proteins from gel to membrane:
Verify efficiency of protein transfer from gel to membrane by silver staining the remaining gel. To improve transfer efficiency 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-c-myc-Peroxidase 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, low expression level, premature translation termination resulting in loss of C-terminal tag sequence:
Include protease inhibitors in lysis buffer. Use alternative expression system or optimize your expression system. Insert multiple tag sequences into target protein to increase avidity of antibody reaction. Use alternative insertion site within the target gene for the epitope tag sequence.

High background, additional bands on blot

Antibody too concentrated:

Decrease concentration of Anti-c-myc-Peroxidase by half.

Wash time too short:

Increase washing time.

Incubation of membrane with substrate too long:

Leave blot membrane in substrate for a shorter time.

Wrong type membrane:
For minimum background, use PVDF membranes for transfer.

Blocking Reagent too dilute:
Use nonfat dry milk (5% w/v) dissolved in PBST as Blocking Solution and antibody diluent. 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.

Signal development time too long:
Reduce development time by half.

Additional bands:
Determine the specificity of the Anti-c-myc-Peroxidase: Include a negative controlcell extract prepared from the host organism and lacking the c-myc-tagged protein.

Materials

     
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