Detection of proteins or DNA on membranes

Chemifluorescent substrates applicable for the ImaGo: ECF Substrate (AttoPhos)

Technical applications

Colony/plaque screening Dot/slot blots Human DNA quantitation (forensics) RFLP/VNTR Southern blotting Western blotting Colony Hybridization Plaque Lifts

Technical Background

Chemifluorescence is the enzymatic conversion of a fluorogenic substrate to a fluorescent product. Fluorogenic compounds (non-fluorescent or weakly fluorescent substances that can be converted into fluorescent products) are available for use with a wide variety of enzymes. ECF reagent kits (Amersham Life Science) simplify chemifluorescence for membrane-based protein and nucleic acid detection. The kits use alkaline phosphatase, which cleaves a phosphate group from a fluorogenic substrate to yield a highly fluorescent product .

Alkaline phosphatase and horseradish peroxidase are currently used interchangeably in most colorimetric and chemiluminescent membrane detection assays, including southern, northern, western, and slot or dot blotting. Chemifluorescence can be used in place of any standard chemiluminescent protocol simply by using an alkaline phosphatase conjugate and the chemifluorescent substrate. Standard buffers and incubations remain unchanged. After a short incubation with the substrate, image the blot on the ImaGo. The light source in the instrument excites the fluorescent product, which emits light in a band centered around 560 nm. The instrument detects this fluorescent signal and creates a digital image of your sample. Genomic Solutions Intelligent Quantifier(TM) software allows rapid and accurate quantitation of the results.

Protein detection.

For protein detection using the Vistra ECF Western Blotting Kit on blots or microplates, the primary antibody is recognized by a secondary antibody that is conjugated to fluorescein. Amplify the signal by incubating the blot with the anti-fluorescein tertiary antibody conjugated to alkaline phosphatase, followed by the substrate. The substrate is cleaved by alkaline phosphatase to yield a fluorescent signal.

The chemifluorescence advantage:

• Specificity and versatility.
  
  Like chemiluminescence, chemifluorescence is based on the affinity binding of an enzyme-conjugated probe to the target molecule. Alkaline phosphatase is readily available conjugated to antibodies that target a wide variety of haptens, or to streptavidin. The use of antibodies ensures specific detection, while allowing great versatility in choice of target molecules for both protein and nucleic acid detection.
  
• Signal amplification.
  
  Under the proper conditions, enzymes continue to convert substrate to product as long as the substrate is available. In chemifluorescence, each alkaline phosphatase conjugate can generate many fluorochrome molecules, continually increasing the signal at any given target. For many samples, only a few seconds of incubation with the substrate yields sufficient signal. However, blots can be incubated for as long as 24 hours to maximize the signal from low-abundance targets.
  
• Nondestructive imaging and adjustable sensitivity.
  
  Unique advantages of chemifluorescence are that imaging a fluorescent blot on a ImaGo fluorescence imaging system is nondestructive, requires no film exposure, and takes only a few seconds. If an initial image is either weak or saturated, adjust the sensitivity of the detection system to maximize the signal over background and then reimage, or continue incubation with the substrate to develop additional signal. With nondestructive imaging and adjustable sensitivity, you can achieve the best results possible for each experiment.
  
• Stability.
  
  In fluorescence, light emission occurs only during exposure to the excitation light (i.e., within approximately 10 nsec of absorption), but a single fluorochrome molecule can be excited repeatedly and can emit light each time. Many fluorochromes are stable for a long time. Fluorescent probes can be kept for many months, and a fluorescent blot can be stored and reimaged after several weeks with little decay in signal. In contrast, a chemiluminescent molecule is autonomously luminous.
  No excitation light is required for chemiluminescence, however the luminescence peaks within minutes of substrate addition and decays over just a few hours.
  
• Limits of detection.
  
  For most blotting applications, the limit of detection of chemifluorescence is equal to that of chemiluminescence, and is better than that of colorimetric detection.
  
• Established protocols.
  
  Chemifluorescence uses the same labeling protocols as chemiluminescence. A chemiluminescence system that already uses alkaline phosphatase can become a chemifluorescent system simply by switching to the chemifluorescent substrate. For a system using horseradish peroxidase (HRP), just replace the HRP conjugate with an analogous alkaline phosphatase conjugate, and incubate with the chemifluorescent substrate. Standard buffers and incubations remain the same.
  
• Quantitation.
  
  Chemiluminescence is linear over about a tenfold range. In an analogous system, chemifluorescence is linear over about a 50-fold range.

Experimental information for Attophos ECF substrate

Advantages:Comparable sensitivity to chemiluminescence
Eliminates the need for dark room, film and chemiluminescent screens
Greater dynamic range than film allows visualization and quantitation of weak and dark bands in the same image.
Substrate for alkaline phosphatase. Converts by enzymatic reaction to fluorescent product, which emits light when excited by a suitable excitation light source.
Alkaline phosphatase-linked secondary antibodies and ECF substrate can be used to amplify signal detection in a variety of microplate and membrane-based applications, such as southern, northern, western, dot and slot blots.
No probe purification step necessary.
Probes stable at least six months at -20 degrees C.


Excitation Maximum:440 nm
Emission Maximum:560 nm

Buffer:2.4M diethanolamine (DEA) in water. 0.23mM MgCl2. pH 10.0 using NaOH. Buffer is stable for one year when stored at 4 degrees C

Method: Use 1mM ECF Substrate dissolved in buffer, or use kitconcentration. For all procedures, including Southerns, westerns and northerns, use clean containers, pipette tips etc. and pipet ECF Substrate onto a overhead projector sheet and lay blot face down onto solution. Be sure to coat blot completely and evenly with substrate.
Incubate as needed, then transfer blot face down to imaging surface.

Always image immediately in case of rapid signal development, then every
15 minutes up to one hour, then every hour, leaving overnight, if necessary.
For western blots on PVDF membranes, blots can be scanned either wet or dry.

Binding:ECF Substrate is dephosphorylated to the fluorescent product, which remains in the region in which it is produced. It is believed to adhere to the membrane by charge interactions.

Detection Limits:Variable depending on the application. 10-10 M of product can be detected in a microplate well.

Linear Range:~1-3 orders of magnitude (highly variable depending on application).

Emission Filters:Attophos filter

Storage:In clean glass or plastic, refrigerated and protected from light.
In buffer (2.4M DEA, pH 10.0, 0.23mM MgCl2)
Diluted reagent at 4 degrees C keeps up to one week.

Handling:Use powder-free gloves to protect ECF Substrate from naturally occurring alkaline phosphatase on hands.

Vendor:Amersham Life Science Inc.

Useful tips for Attophos

• Always image immediately. Some blots can develop within minutes.
• Use of water between imaging tableglass and plastic bag reduces optical refraction and results in a clearer image. Use Mylar rather than Saran wrap as a cover. It is far less fluorescent.
• Covered refrigerated solution can be used up to one month.
• Use of containers and tips cleaned by sterilizing or by rinsing with 0.01M HCl or boiling water reduces background resulting from ambient alkaline phosphatase.
• Autoclaving of blocking solution and filtering of reagents can decrease background from ambient alkaline phosphatase.
• Use of powder-free gloves is important, as the powder fluoresces.