Simplified Detection of Proteins in the Picogram and Femtogram Range

Western blotting is one of the most commonly used applications in protein research. Recent advances in chemiluminescent substrate design have meant that chemiluminescent blots scanned using charge-coupled device (CCD) image analysis systems can generate real-time images in which smaller amounts of protein can be detected than on blots labeled with traditional colorimetric or fluorescent dyes.

Another advantage of using chemiluminescent substrates is that the blots can be stripped and reprobed, which saves scientists time running gels, as well as time transferring proteins from gels to blots. The disadvantage of using chemiluminescent substrates is that substrates can produce background noise if left for long exposure times, especially when using film for capturing blot images. Additionally, either a darkroom and film or an imaging system is required to visualize results. Even an imaging system, which captures the optimum images of chemiluminescent blots, can be daunting since so many variables need to be set to detect bands of protein in the picogram and femtogram range.

Since chemiluminescence is still the best choice for scientists who need to see very small amounts of proteins and reprobe the blot with many different antibodies, this article discusses the issues of background noise and configuring an imager to achieve optimum imaging results of picogram and femtogram amounts of proteins.

System for imaging chemiluminescent Western blots

Figure 1 - G:BOX iChemi XR image analyzer.

The G:BOX iChemi intelligent chemiluminescent imaging system (Syngene, Frederick, MD) was developed to overcome the difficulties in capturing perfectly exposed chemiluminescent blots (Figure 1). Unlike many chemiluminescence imaging systems, the processor of the G:BOX iChemi automatically assesses the sample, sets its own exposure time and sensitivity, and autoscales the captured image. With these features, users can produce high-quality images with very good contrast, without any of the guesswork or trial-and-error imaging usually associated with capturing images of chemiluminescent blots.

To demonstrate its imaging range, the G:BOX iChemi system was used to image LumiGLO® and LumiGLO Reserve™ peroxidase chemiluminescent substrates (KPL, Gaithersburg, MD). LumiGLO detects low-picogram quantities of target protein on blots with signal linearity across a broad dynamic range of detection, with a high signal-to-noise profile. LumiGLO Reserve allows detection levels as low as the femtogram range, with little nonspecific background signal. While many different substrates may be used with the image analyzer, LumiGLO substrates can provide researchers with optimal sensitivity and S/N when using the G:BOX iChemi XR system.

Method

Generating chemiluminescent labeled Western blots

Five sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were run. Two were loaded with five dilutions of human IgG (Lampire Biological Laboratories, Pipersville, PA) (500 ng, 50 ng, 5 ng, 500 pg, and 50 pg), and three were loaded with five dilutions of human IgG (5 ng, 500 pg, 50 pg, 500 fg, and 50 fg). Each gel was also loaded with a set of prestained protein markers (Blue Pre-Stained Protein Molecular Weight Marker, Thermo Fisher Scientific, Rockford, IL). The proteins were transferred from acrylamide gels onto nitrocellulose membranes using a standard electroblotting method. All the membranes were incubated overnight in 1× Detector Block solution (KPL) with 1% Detector Block powder. The blots were incubated for 1 hr at 25 ºC in 1× Detector Block containing a horseradish peroxidase (HRP) goat antihuman IgG (KPL) diluted 1:10,000. The membranes were washed in 1× Wash Solution (KPL). LumiGLO was applied to one blot for 1 min at 25 ºC; substrate A was applied to the second blot for 5 min at 25 ºC; and substrates B, C, and LumiGLO Reserve were applied to the third, fourth, and fifth blots, respectively, for 1 min, all according to the manufacturers’ instructions.

Imaging chemiluminescent blots

Each membrane was placed inside the G:BOX iChemi XR darkroom. While the darkroom door was open, the focus was adjusted using the colored, prestained protein markers as a focusing guide. The system was then set up without any light source, since excitation and emission are not necessary with chemiluminescent substrates and without any filters present. The system’s GeneSnap software was programmed to “capture series” at 60-sec intervals for 15 min. The G:BOX iChemi XR then automatically selected and displayed the image of each of the five blots, which showed the correct exposure with minimum background noise after 1-min and 15-min exposure times.

Results

Figure 2 - LumiGLO (left) and substrate A (right) labeled Western blot images generated by G:BOX iChemi XR after a 1-min exposure showing, from left to right: 500 ng, 50 ng, 5 ng, 500 pg, and 50 pg of human IgG (Figures 2–5 kindly provided by KPL).

The image produced by the G:BOX iChemi XR of membranes 1 and 2 (Figure 2) shows that three bands (500, 50, and 5 ng) are visible after a 1-min exposure with substrate A, and four bands (500 ng, 50 ng, 5 ng, and 500 pg) are visible with LumiGLO.

Figure 3 - LumiGLO (left) and substrate A (right) labeled Western blot image captured by the G:BOX iChemi XR after a 15-min exposure showing, from left to right: 500 ng, 50 ng, 5 ng, 500 pg, and 50 pg of human IgG.

The image of membranes 1 and 2 (Figure 3) generated by the G:BOX Chemi XR shows that five bands (500 ng, 50 ng, 5 ng, 500 pg, and 50 pg) are visible with both LumiGLO and substrate A after a 15-min exposure. Therefore, both substrates will detect human IgG protein in the 50-pg range if left for a sufficient exposure time, which is consistent with the levels of protein detection claimed by the manufacturers.

Figure 4 - LumiGLO Reserve (left), substrate B (middle), and substrate C (right) labeled Western blot image generated by G:BOX iChemi XR after a 1-min exposure showing, from right to left: 5 ng, 500 pg, 50 pg, 500 fg, and 50 fg.

The image produced by the G:BOX iChemi XR of membranes 3, 4, and 5 (Figure 4) shows that four bands (5 ng, 500 pg, 50 pg, and 500 fg) are visible with LumiGLO Reserve, while three bands (5 ng, 500 pg, and 50 pg) are each visible with substrate B and substrate C after a 1-min exposure.

Figure 5 - LumiGLO Reserve (left), substrate B (middle), and substrate C (right) labeled Western blot image captured by the G:BOX iChemi XR after a 15-min exposure showing, from right to left: 5 g, 500 pg, 50 pg, 5 pg, and 500 fg.

The image of membranes 3, 4, and 5 (Figure 5) generated by the G:BOX iChemi XR after a 15-min exposure shows that five bands (5 ng, 500 pg, 50 pg, 500 fg, and 50 fg) are visible with LumiGLO Reserve and substrate C, while four bands (5 ng, 500 pg, 50 pg, and 500 fg) are visible with substrate B. All three substrates will therefore detect human IgG protein in the 500–50 fg range if left for a sufficient exposure time, consistent with the levels of protein detection claimed by the manufacturers.

A summary of the performance of the five different chemiluminescent substrates with the G:BOX iChemi XR is shown in Table 1.

Table 1 - Comparison of chemiluminescent substrate detection with G:BOX iChemi XR system

Conclusion

The G:BOX iChemi XR system provides an easy method of accurately detecting proteins with a range of different chemiluminescent substrates. LumiGLO and LumiGLO Reserve substrates provide better sensitivity and linearity than other commonly used chemiluminescent substrates. Since these systems are designed for fully automated setup and walkaway image capture, scientists can use substrates such as LumiGLO and LumiGLO Reserve for rapid, low-picogram- and femtogram-level detection. Alternatively, they can leave their exposures for longer periods using the G:BOX iChemi system with dyes such as substrates A, B, and C to produce optimum results in the picogram and femtogram range.

Ms. Maia is Vice President of Sales, Syngene, 5108 Pegasus Ct., Ste. M, Frederick, MD 21704, U.S.A.; tel.: 301-662-2863; fax: 301-631-3977; e-mail: [email protected].  Ms. Webster is Technical Services Supervisor, and Dr. Harkins is Senior Scientist, R&D Manager, KPL, Inc., 910 Clopper Rd., Gaithersburg, MD, U.S.A.