The Western Blot – a tried and true experimental protocol where protein structures are separated via molecular weight/charge and transferred to a membrane before visualization by a chemiluminescent solution (say that three times fast!). Seems simple, right? While the step-by-step process of a western blot has for the most part remained the same over the years, variations in solutions, procedures and reagents may increase the efficacy of your results. For example, when it comes to choosing a membrane for protein transfer there are good arguments for choosing between a PVDF and Nitrocellulose. Which one suits your protein sample best?
|
PVDF |
Nitrocellulose |
Protein Size |
Better for high MW proteins |
Better for mid to low MW proteins |
Sensitivity |
Higher protein binding capacity and sensitivity |
Strong binding capacity, not as suitable for low expression level targets |
Strip & Re-probe |
Performs well |
Possible, but can lose sensitivity in the process |
Durability |
More durable |
Less durable |
Background Noise |
Slightly higher signal |
Low background signal |
Saturation |
Requires methanol or ethanol prior to transfer |
Requires methanol during transfer – may lead to smaller membrane pore size and precipitation of larger proteins |
Other uses |
Southern, northern and dot blots – amino acid analysis and protein sequencing |
Most commonly used for WB, Southern blot and northern blot |
For more information on how to select a membrane for western blot, visit pages 16 and 17 of Novus Biologicals Western Blot Handbook. Additionally, Novus Biologicals carries ready to use PVDF membranes pre-set with human tissues for efficiency and reliability! Don't forget to use a loading control when conducting a western blot experiment; take a peek at our actin antibodies, alpha tubulin antibodies, beta tubulin antibodies and GAPDH antibodies to find one that works best for your application.
