Gel shift assays or electrophoretic mobility shift assays (EMSA) provide a simple method to study DNA:protein interactions. This assay is based on the principle that a DNA-protein complex will have different mobility during electrophoresis than non-bound DNA. These shifts can be visualized on a native acrylamide gel using labeled DNA to form the DNA-protein binding complex.
Figure 1. EMSA performed with IRDye 700 AP-1 oligos. Reprinted with permission from Electrophoretic Mobility Shift Assay (EMSA) Using IRDye Oligonucleotides.
And when you are ready to image, there is no need to remove the gel from the glass plates. This makes gel handling easier and allows running the gel further, if needed, after scanning is completed. Possible deformations or tearing of the gel while separating plates are also eliminated.
For more information, refer to our Technical Note on Infrared EMSA detection.
Introducing the NEW! Quick Western Kit – IRDye® 680RD
The Quick Western Kit – IRDye® 680RD provides a universal detection reagent that can be combined with the primary antibody incubation step, eliminating the need for a secondary antibody incubation step.
Does not require a separate primary antibody labeling step, saving time and antibody
Faster method of detection compared to the traditional 2-step method, which can take up to 4 hours
Reduces the total Western blot procedure by at least 90 min
The kit can be used to detect primary antibodies from a variety of hosts and has been shown to recognize primary antibodies to recombinant tagged proteins (i.e. 6X His, Myc, DDK, etc.)
IRDye 680RD Detection Reagent is known to have high specificity for IgG from:
The IRDye 680RD Detection Reagent does not work with:
The Detection Reagent is known to have lower specificity for rat, horse, and hamster. The Detection Reagent has been specifically tested and qualified for Western blot applications. If additional specificity and/or affinity are required, please use IRDye conjugated secondary antibodies for detection.
Two-fold dilutions of crude lysate containing an overexpressed 6X-His tagged DNA polymerase was loaded in Lanes 3-7. A Histag molecular weight marker (Invitrogen) was loaded in Lanes 1 & 9. The nitrocellulose membrane was blocked with Odyssey Blocking Buffer (PBS) and probed with anti-His Tag Rabbit Polyclonal Antibody (GenScript; 1:500) and IRDye 680RD Detection Reagent (1:1000) for 1 hour. The image was collected on the Odyssey CLx.
PSVue® 794 is a near-infrared fluorescent probe for detection of apoptotic and necrotic cells, bacteria, and other anionic membranes. The compound exhibits fluorescence excitation maximum at 794nm and emission maximum at 810 nm and through its zinc(II)-dipicolylamine (Zn-DPA) moiety, it has been found to bind strongly to negatively charged bacterial cell walls (e.g. S. aureus, E. coli) and necrotic regions present in various tumors (e.g. mammary, prostate, glioma) in vitro and in vivo. In particular, it has also been found to bind to the phosphatidylserine (PS) residues exposed on the cell surface of apoptotic cells, making it a more cost-effective alternative to fluorescently-labeled Annexin V in various cell death assays.
Figure 1. MPTP was used to induce cell death in mouse brains as a model for Parkinson’s Disease. C57BI/6 mice were treated with MPTP to selectively destroy dopaminergic neurons. Mice were then injected with PSVue dye or control dye and imaged on the Pearl® Imager 68 hrs post injection. A. control (i.e. non-targeting) dye; B. and C. PSVue dye; D. excised brains from the three animals.
Download a scientific poster presenting information on the use of PSvue 794 in studying Alzheimer’s Diesase, Parkinson’s Diesase, and contact dermatitis in mouse models.
As you can see in the images below, the reactivity of secondary antibodies ranges widely between vendors, even within the same species and especially between host species. The ratio of HRP enzyme to antibody varies and may affect the detection of the target. Try secondary antibodies from several vendors to find the ones that give the most satisfying data.
Serial dilutions of mouse or rabbit IgG were spotted onto nitrocellulose (2500 pg to 0.3 pg) and probed with HRP-conjugated secondary antibodies from various vendors. Blots were detected with SuperSignal® West Dura chemiluminscent substrate (Thermo Scientific) and exposed to film for 15 seconds.
Another one of those Notes: When evaluating the performance of the primary and secondary antibodies, try different blocking buffers (yes, we had a post on that too – 8-Dec-11 - The Best Offense is a Good Blocker), as the choice of blocker can affect the antibodies’ performance.
For optimal results do not dilute the HRP-conjugated secondary antibodies with blocking buffer containing sodium azide as a preservative (e.g., Odyssey® Blocking Buffer), as it will inhibit peroxidase activity and result in less light production.