Go Green with Infrared Imaging:
Cleaner Planet, Better Data
No Darkroom, No Harsh Chemicals
In addition to the environmental advantages of infrared imaging, it has also become the industry leader in Western blot technology since LI-COR pioneered the field more than 10 years ago. When used with corresponding infrared fluorescent dye-labeled secondary antibodies, LI-COR infrared technology offers the following benefits:
- Quantitative analysis and a wide linear dynamic range that is not available with traditional chemiluminescent methods
- Detect strong and weak bands on the same blot, without "blown out" lanes or hidden bands (Fig. 1)
- Detect two targets simultaneously on the same membrane to increase quantification accuracy
- Infrared dyes offer advanced signal stability that allows for convenient and reproducible data that are not time-sensitive - data are not contingent on the lifespan of an enzymatic reaction (Fig. 2)
Consider the following characteristics associated with film development and its required reagents:
- Photo solutions have high chemical and biological oxygen demand (COD and BOD) and are often treated with , peroxide, or aeration to reduce COD.
- Photo chemicals use non-biodegradable compounds in developers and washing aid solutions, such as EDTA, DTPA, NTA, and borate. Water containing these chelating agents can leach metal from water treatment equipment, as well as pipes.
- Darkrooms use potassium ferricyanide, potassium dichromate, or permanganate for bleaching. These compounds decompose in the wastewater streams and are highly regulated for sewer disposal, at a dilution of 20,000 times or more.
- Hydroxylated benzene compounds are commonly used as developing agents. They are harmful to humans and experimental small animals, and have been noted as mutagenic. These compounds have a high COD.
Figure 2 [Right] Western blots containing purified transferrin were processed using chemiluminescence (10 ng - 1.2 pg) or near-infrared detection (40 ng - 4.8 ng). Panel A shows the loss of signal for chemiluminescence detection after 60 minutes on the Odyssey Fc. Panel B shows the stability of a Western blot processed with IRDye 800CW secondary antibody after 18 months imaged on the Odyssey Classic. The lower limit of detection is maintained in the near-infrared Western blot with all of the data points available (14 points total) even after storage for 18 months. Compare this to the chemiluminescence detection where 7 of the same data points are detected after
Figure 1. Serial dilutions (10 ng to > 1 pg) of purified human transferrin (Tf) were used to assess Western sensitivity. The Odyssey Classic using infrared fluorescence detection, reproducibly detected 1.2 pg of Tf, while only 4.9 - 9.5 pg was detected with chemiluminescence. Infrared detection sensitivity was approximately 200-fold greater than previous studies with visible fluorophores (Cy®3, Cy®5, or FITC).
