Question: I have received an antibody which has not been used before in Western blot, what protocol should I use? 

To save time, before doing any experiments, it is recommended to conduct a short theoretical analysis and answer the following questions: 

  • Is the antigen sequence used to elicit this antibody also conserved in the protein I am aiming to detect?
  • Is the antibody made to native or denatured protein
  • Is the protein I am aiming to detect abundant or of low expression
  • If the protein is lowly expressed, using a specific cellular fraction will concentrate the target protein to detectable levels. 
  • If the protein is lowly expressed and cytoplasmic, TCA/acetone precipitation is recommended. 
  • What is the expression on a tissue level? Should a specific tissue, or its fragment be used?
  • Is the protein stable, or can it be subjected to degradation during protein extraction
  • Is the protein light-sensitive? Should protein extraction be conducted in a darkroom? What extraction buffer to use?
  • What is the MW of the protein? If of low molecular weight, gel electrophoresis and blotting conditions have to be adjusted. Recommended conditions can be found here.
This analysis will ensure that correct conditions are applied, which will secure that enough of the target protein is transferred to the membrane, which will facilitate antibody binding. 

 Samples: WT, specific mutant
Protein load/well: >50 µg
Blocking: 5% non-fat milk, 1 h/RT
Primary antibody: 1: 500 or 1: 1000, ON/4°C
Secondary antibody: 1: 25 000, 1 h/RT
Record results

Including samples from knockout or knockdown mutants is crucial, if the antibody is used for the first time. This will confirm its binding to the correct target protein. Using other controls at this stage, as for example overexpressed protein or actin, is not relevant. Without a mutant sample, we will not be able to confirm tha the antibody is binding to the correct protein. If it is not possible to obtain vital mutants, there are indirect method to confirm binding of the antibody to the target protein under investigation. 

To avoid false positive results with antibodies, the used set of samples have to be chosen carefully.