This experiment uses a robust reproducible fluorescence based assay to determine exonuclease activity. Incubate the purified enzyme with the fluorescent DNA substrate to allow the enzyme to sequentially degrade DNA. Then resolve the DNA on an acrylamide urea gel to separate the degradation products by size.
Next, use fluorescence imaging to identify and quantify the extent of exonuclease activity. Analyzed results can determine the preferred substrate reaction conditions, processive and overall activity of the enzyme. The main advantage of this technique over existing radioactivity based techniques is that the DNA substrates are stable for a long time, allowing excellent reproducibility, decreasing cost, and increasing safety.
First, adjust the bacterial lysate to 20 millimolar ole, using an appropriate volume of two molar emit ole stock solution. Then adjust the volume to at least one milliliter using 20 millimolar ole wash, A one milliliter hist trap column with 10 milliliters of milli Q water. Then equilibrate the column with 10 milliliters of 20 millimolar ole.
Next, load the lysate onto the column as the lysate is loaded onto the column, significant back pressure may be experienced. This is perfectly normal. Do not be tempted to apply too much force to the syringe and maintain a steady even pressure.
It is usual to see a mild color change as the lysate enters the column. Collect the flow through in a sterile biju bottle seven milliliter capacity and store on ice. Then wash the column with 10 milliliters of 20 millimolar emit ole.
Now pass five milliliters of 40 millimolar iole through the column. Watch the plunger of the syringe carefully to measure the one milliliter fractions that are collected into each of the micro fuge tubes. Similarly sequentially, pass five milliliters of each iole concentration through and collect five one milliliter fractions each time.
Assay the fractions by dot blotting and probing with an HRP conjugated anti hiss antibody. To identify the peak fractions. Keep all reagents at four degrees Celsius aliquot the master mix and a sterile DNA free 0.5 milliliter Micro fuge tubes also include a no enzyme control to visualize the initial substrate.
Now set a heat block to the appropriate temperature for enzyme activity. Prepare a IDE containing stop solution and store it at room temperature. Organize the workspace near the heat block, including the final reagents to be added to the reaction.
Mix a one to 10 microliter pipetter in tips, a timer, a cryo rack, or an ice bucket and stop solution. Start the timer on addition of enzyme to the first tube and place the tube in the heat block. Continue adding enzyme to the rest of the tubes at 15 second intervals, and place the tubes in the heat block as the timer reaches the correct time.
Add 10 microliters of stop solution to the first sample and mix by pipetting Place immediately on ice. Continue to stop the reactions at the appropriate time intervals and in the same order as they were set up so that each reaction has had exactly the same incubation time load half or all of each sample onto an acrylamide gel load. One x stop solution containing dye in one or more of the lanes to ascertain how the gel is running.
After electrophoresis, remove the spacers and one gel plate. Carefully rinse and wipe the gel plate thoroughly to remove traces of precipitated urea. Set the plate with gel down onto the glass space of the gel holder and place it into the imager if necessary.
Note the coordinates of the gel on the glass. Open the appropriate software and scan the noted region of the scanning area using the conditions detailed in the text protocol. Crop the image and compress the data.
If needed, save then export as a file for analysis. Performing in vitro analysis of exonuclease activity requires a number of preparatory steps. In addition to the actual analysis for these fluorescence based exonuclease assays, it is critical to optimize detection of the fluorescence imaging system.
For example, an oligonucleotide substrate that is fluorescein labeled requires a filter that permits excitation at 470 nanometers and emission at 520 nanometers. Using this filter, it is possible to increase the ability to detect low concentrations of substrate or product by adjusting both the sensitivity and resolution of the imager. Note, there is some tolerance in terms of filter bandwidth.
As a suboptimal filter choice still allows partial detection of the substrate, though with much lower sensitivity. An appropriate combination of labeled oligonucleotide concentration and imager settings can result in robust detection of the substrate. This is important since the degradation products upon incubation with an active exonuclease will be present at lower amounts in each band as the substrate has been sequentially fragmented.
A successful exonuclease assay will cause degradation of the substrate sequentially towards the flora. Four, resulting in a characteristic ladder like pattern of DNA on the gel representative of exonuclease degradation. Once optimized for detection, the assay can be used to detect differential activities both qualitatively and quantitatively.
For example, the presence or absence of activity can be determined for nuclease mutants compared with wild type protein. Different substrates may also be tested for their ability to be degraded by the nuclease. Under investigation.
This in vitro assay can be used to test substrate specificity and process of a nuclease of interest.
