Western Lightning Chemiluminescence Reagent Plus From Revvity

Western blotting is a standard laboratory technique routinely used in molecular biology; we use it specifically in our lab to test the expression levels of amyloidogenic disease proteins and transfected antibodies in cellular and animal models of neurodegenerative diseases. After lysis and separation of cell debris by centrifugation, approximately 25 ìg samples of tissue or cell lysates' protein are separated by SDS-Polyacrylamide Gel Electrophoresis, transferred to polyvinylidene difluoride (PVDF) membranes and then probed with a primary antibody specific to the protein of interest. In Revvity's Western Lightning Chemiluminescence Reagent Plus system, secondary antibodies coupled to the enzyme horseradish peroxidase (HRP) are then used to detect the presence of the protein by catalyzing the emission of light at a wavelength of 428 nm from the oxidation of enhanced luminol reagent added in the presence of an oxidizing reagent.

The actual process is very simple. After extensive washing with TBS/Tween buffer between antibody incubations (about 4-5 washes of 15 mins each), the membrane is lightly blotted against tissue paper to drain excess buffer and then incubated with a mixture of equal volumes (1 ml each of the Enhanced Luminol Reagent and the Oxidizing Reagent) for about a minute. The blot is then enveloped in Saran Wrap smoothing out air pockets, and quickly positioned in a film cassette, protein side-up, for exposure to X-ray film in a darkroom. The film can be exposed from 3 seconds to 10 minutes, according to the levels of protein to be detected.

The strongest advantage of this system in my estimation is that there are a minimum of reagents required to work with, hence reducing possibilities of error. No pre-equilibration of reagents are required, and it can be used with HRP-conjugated antibodies from other companies. The reagents are non-radioactive; the system is quick (less than 5 minutes to apply and incubate) and easy to use. After probing and analysis, the membrane can then be stripped and reprobed with a second set of antibodies. Although the product has a long shelf-life (1 yr) the product must be stored at 2-4°C. Reagents need to be pre-mixed in correct ratios for optimum performance, and membranes need to be efficiently drained to prevent a diluting effect of wash buffer on oxidizing reaction. In my experience, this product usually gives good results; however, I have noticed that the time required for equivalent exposure results varies from blot to blot as overexposure is observed sometimes at 1 minute, at other times as little as 20 seconds, all else being held equal.

In summary, this system works well at first, but the performance of the reagents deteriorates as shelf life is approached.