1. Check the loading of the samples. The amount of protein loaded into each lane of the gel should be equal in order to ensure that the results are comparable. This can be done by staining the gel with a protein stain, such as Coomassie Blue or Amido Black. 
  2. Check the molecular weight markers. The molecular weight markers should be evenly spaced and should run at the expected sizes. This can be done by comparing the sizes of the bands on the marker to known standards. 
  3. Check the specificity of the antibodies. The antibodies used in the western blot should be specific for the protein of interest. This can be done by comparing the bands on the blot to a known positive control sample. 
  4. Quantify the bands. The intensity of the bands can be quantified using a densitometer or by using image analysis software. This can be used to compare the levels of the protein of interest in different samples. 
  5. Consider the experimental conditions. The results of a western blot can be affected by a number of factors, such as the type of gel used, the running conditions, and the concentration of the antibodies. It is important to consider all of these factors when interpreting the results of a western blot. 
  6. It is important that the protocol is reproducible. Using a reliable automated system to handle all post transfer steps: blocking, washing, primary/secondary antibody application steps. The BlotCycler Touch or Mini are ideal for producing consistent publication quality results. 

 

A few other points to consider: 

  • Bands that are not present in the control sample but are present in the experimental sample may indicate that the protein of interest is up-regulated in the experimental sample. 
  • Bands that are present in the control sample but are not present in the experimental sample may indicate that the protein of interest is down-regulated in the experimental sample. 
  • Changes in the size or shape of bands may indicate that the protein of interest has been modified, such as by phosphorylation or glycosylation. 
  • The absence of a band for a protein that is expected to be present may indicate that there is a problem with the western blot, such as a lack of protein in the sample or an antibody that is not specific for the protein of interest.