Why is the actual Western blot band size different from the predicted?

Western blotting is a technique that separates proteins based on size. In general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:

• Post-translational modification - e.g. phosphorylation, glycosylation etc, which increases the size of the protein
  
• Post-translation cleavage - e.g. many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases
  
• Splice variants - alternative splicing may create different sized proteins produced from the same gene
  
• Relative charge - the composition of amino acids (charged vs non-charged)
  
• Multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands
  

ONE HOUR WESTERN BLOT!!

NO ONE SPEND 4-5 HOURS FOR WESTERN BLOT ANYMORE

The western enhancer was designed to help you get your Western blot result quick and easily. 
It only takes 1-1.5 hour to replace the 4-5 hours or overnight process of the classical western blotting.

Advantages:

1. The rapid process: the kit was designed to take one hour (1/2 hour for primary, 1/2 hour for secondary antibody binding and 8 min. washing to obtain a desired Western blot data.

2. The time saved: It takes only 1-1.5 hour instead of the 4-5  hours classic western process,  with this kit, you can efficiently probe and re-probe membrane for Western, and Dot blots with multiple antibodies or with multiple concentration of an antibody for a desired result.

3. The money saving: One-Hour Western (OHW) is designed to increase the affinity of antibody to target antigen by 6-10 times in comparison to the classical Western method. And the used antibody in OHW antibody binding solution can be kept at 4˚C for 4-6 weeks.

Fig.1,Mouse tissue protein, used the actin 1: 30,000.

Fig.2-A, Mouse tissue protein, the antibody –β-Catenin,1ug/ml, 2 % Milk-HHWE, the result shows the high concentration antibody used. 

Fig.2-B, The good result was obtained from the same membrane-Fig.2 A,re-probed with Anti-β-catenin (0.1ug/ml) in 2% milk-HHWE.                                

Fig.3,Human cell line protein, antibody-NFкB p50, E-10 (Santa Cruz, Cat#, sc-8414),0.66ug/ml in 2% Milk-HHWE.  

 

Ref: Link 1
       Link 2