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Western Blot FAQs

What protocol was used to perform the blot shown in the data sheet of the Bethyl antibodies?

All of Bethyl’s antibodies are tested using the reagents from the ReliaBLOT® kit. A ReliaBLOT protocol and western blot protocol can be found in the Protocols Section of the Bethyl website.

If I don’t use the ReliaBLOT® kit, what blocking buffer should I use?

The recipe for ReliaBLOT® blocking buffer is proprietary. An acceptable substitute would be 5% milk (Carnation, non-fat, dry)/TBS/0.5% tween. A protocol for Western Blotting can be found in the Protocols Section of the Bethyl website.

Can you give me the exact sequence of the peptide used as the immunogen to generate the antibody?

The sequences of the immunogens are proprietary. All of the data sheets on our polyclonal antibodies provide a range of amino acids in the protein to which the peptide maps.

Why is my band/signal not migrating at the same size that you show in your data sheet?

  1. The migration patterns of molecular weight markers can differ between vendors.
  2. The type of gel and buffer system used can have effects on protein mobility.

Why do some proteins not migrate at the predicted molecular weight?

  1. Proteins that are highly basic or acidic (charged) may not migrate at their theoretical molecular weight.
  2. Sometimes proteins will retain some secondary structure (re-folding) which can alter mobility.
  3. Post-translational modifications will alter protein mobility.