Test your knowledge of protein blotting membranes

January 10, 2012 at 1:42 pm Leave a comment

When performing western blotting, we often spend lots of time contemplating our protein sample preparation, the ideal percent acrylamide gel to use or our primary or secondary antibody selection. However, we tend not to spend too much time thinking about which membrane we should be using or how our handling of that membrane may affect the outcome of our experiment.

There are a variety of membrane types available for western blotting, each offering key attributes to suit particular experimental conditions. Below is a guide to protein blotting membranes along with some of their uses and limitations (click on the table for a clearer image).

Membranes are typically available in 0.45um and 0.2um pore sizes with the former recommended for most analytical blotting experiments and the latter more suitable for transfer of low molecular weight proteins.

Nitrocellulose is probably the most common membrane. Key features include:

  • irreversible protein binding
  • instantaneous protein binding
  • quantitative to 80-100ug/cm2
  • easily wetted in water or transfer buffer
  • compatible with a wide range of protein detection systems
  • fragile
  • not recommended for stripping and reprobing
  • Supported nitrocellulose withstands reprobing and autoclaving

Polyvinylidene Difluoride (PVDF) membrane is an ideal support for N-terminal sequencing, amino acid analysis and immunoassays of blotted proteins. The following applies to PVDF membranes:

  • PVDF retains protein during exposure to acidic or basic conditions and in the presence of organic solvents
  • good for use with rare, low abundance prteins due to high protein retention
  • high binding efficiency in presence of SDS
  • must be wetted in 100% methanol prior to use
  • does not require use of methanol in transfer buffer

Immun-Blot PVDF membranes are hybrid membranes that exhibit the high protein binding efficiency of PVDF without the associated nonspecific protein binding.

The above information was taken from Bio-Rad Laboratories Protein Blotting Guide. For more information download the guide or visit Bio-Rad’s protein membrane website.

Entry filed under: Bio-Rad Tech Note, protein blotting, Proteomic Technotes, Western Blot and ELISA. Tags: , , , .

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