Highly sensitive protein identification using Azure Imaging System models 280, 300, 400, 500, or 600.
Simultaneous imaging of chemiluminescent samples and colored molecular weight markers. Images are captured serially, and then overlaid within the capture software.
Chemiluminescent Western blots are popular assays for assessing protein expression. In this indirect detection method, chemiluminescent substrates emit light when reacted with an antibody conjugated to an enzyme. This is an easy-to-follow chemiluminescent Western blot protocol with full list of materials and reagents, technique and results to guide you through your experiment.
Material / Reagent
SDS-PAGE Gel Electrophoresis System
Transfer Cell System
Molecular Weight Protein Ladder
Plastic (not metal) forceps strongly recommended
Rotary or Rocking Platform
Rocking is recommended over orbital shaking
Chemiluminescent substrates allow for the identification of proteins of interest using specific antibodies following Western blotting membrane transfer.
Western blotting is a technique in which proteins are first separated by size through polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to a membrane. From there, the membrane can be treated with chemiluminescent detection in order to visualize a specific protein of interest.
Chemiluminescent detection is a popular method for Western blotting that utilizes an enzyme-substrate reaction that produces light. Two commonly used chemiluminescent enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP) with one major difference being HRP’s higher sensitivity than AP. Historically, x-ray film was used to visualize chemiluminescent signals, but advancements in imaging technology have allowed CCD-based imaging systems like the Azure Imaging System to capture these signals without the inconveniences of film development.
Highly sensitive identification of a specific protein of interest using a primary antibody directed against the target.
General guidelines are provided as a reference for experiment-dependent protocol optimizations.
1. Sample Preparation
Note: Sample preparation
method will differ
on the experiment.
a. Homogenize cell cultures while keeping cells at ice-cold temperatures to prevent
b. Add ice-cold lysis buffer containing a protease inhibitor cocktail, as well as phosphatase inhibitor if working with phosphorylated targets to homogenized cells. Centrifuge to separate lysate supernatant from cell debris and collect the lysate supernatant only.
d. Add sample buffer to the lysate supernatant. Ideally, prepare lysate stocks that are at a final protein concentration of at least 1mg/mL. If reduction of disulfide bonds is desired, include a reducing agent such as DTT, β-mercaptoethanol or TCEP in the sample buffer.
e. Denature the sample by heating at 98°C for 5 minutes.
2. SDS-PAGE Gel Electrophoresis
a. Unpack the pre-cast SDS PAGE gel by unwrapping the gel, removing the tape at the bottom of the gel, and slowly removing the plastic comb from the gel lanes.
b. Place the unwrapped gel with the well’s open side facing inward on the Azure Aqua Quad Mini-Cell’s gel holder. If an odd number of gels are being run, use a plastic barrier to ensure each gel holder can contain liquid. Push the sides of the gel holder inwards to secure the gels and barriers into place.
c. Fill the middle of each gel holder with 1x Tris-Glycine SDS PAGE Running Buffer and check for leaks. If buffer leaks out of the bottom of the gel holder, readjust the sides of the gel holder to ensure that both gels or barriers are sealed securely.
d. Once the middle of each gel holder is filled to the top, fill the body of the Azure Aqua with 1x Running Buffer up to either the “2 Gel” or “4 Gel” lines based on the number of gels being run, rounded up to the nearest multiple of two.
e. Once the gels are secured and the Aqua filled with running buffer, load the 2.e denatured/reduced samples and the molecular weight marker into the gel lane.
f. Set up the Azure Aqua Quad Mini-Cell by connecting the red and black power cable from the lid to the Azure Aqua Power Supply. Place the lid on the Quad Mini-Cell by securely attaching the color-coordinated electrodes to the correct colors on the gel holder. Make sure the color of the electrodes on the gel holder matches the labels on the side of the Quad Mini-Cell as well.
g. After connecting the electronics securely, turn on the Azure Aqua Power Supply with the switch on the back-right of the instrument.
3. Membrane Transfer
Note: The following process will only be detailed for one gel.
a. Gather one blot incubation tray, one transfer membrane, four blotting papers, plastic tweezers, a container large enough to fully submerse the stack in buffer, gel roller, and one transfer cassette from the red-and-black transfer case within the Azure Aqua Transfer Cell.
b. Equilibrate the PVDF membrane in 1X Azure Transfer Buffer. First, place enough 200 proof Methanol in a blot incubation tray to completely submerge the blot.
Gently agitate for 15 seconds to ensure that the membrane is completely wet. Next, decant the methanol and add ~50mL of high purity water to the membrane. Incubate with rocking for 5 minutes. Decant the water and add ~25mL of 1X Azure Transfer Buffer to the membrane. Incubate with rocking for at least 5 minutes.
c. Open the transfer cassette by sliding the white lock along its track and rotating upwards. Separate the two porous sponges within and place them on opposite sides in a large container containing 1X Azure Transfer Buffer.
d. Using the forceps, fully immerse four blotting papers in transfer buffer and place two on each sponge within the transfer cassette.
e. Remove the gel from its casing by cracking open the casing with a designated metal or hard plastic tool.
• The very top and bottom of the gel should be cut clean off to make handling and removal easier. Be sure not to cut so much that any of the sample will be removed.
f. Place the gel centered on the soaked blotting paper that is on the black side of the transfer cassette.
g. Place the PVDF membrane that has been equilibrated in 1X Azure Transfer Buffer onto the gel. Ensure that the gel and membrane are exactly the same size and aligned for the best transfer.
h. Close the transfer cassette so that, from the black side to the clear side, the order should now be: outer sponge, soaked blotting papers, SDS-PAGE gel, equilibrated transfer membrane, soaked blotting papers, outer sponge.
i. Place the transfer cassette back into the red-and-black transfer case in the Azure
Aqua Transfer Cell such that the clear side of the cassette is towards the red side of the case. Additionally, place a frozen ice pack into the Transfer Cell to maintain a low temperature during transfer.
j. Fill the Azure Aqua Transfer Cell with ice-cold Transfer Buffer to the top line labeled “Blotting” and connect it to the Azure Aqua Power Supply.
• Securely fasten the lid of the Aqua Transfer Cell with the red and black electrodes matching their respective colors on the red-and-black transfer case.
• Plug the power cable into the Azure Aqua Power Supply with the matching colors.
k. Set the power supply to 55V and run for 35 minutes.
l. After the transfer process is finished, unplug the Aqua Transfer Cell from the power supply and turn the power supply off.
m. Remove the transfer cassette and open it. Using the forceps, carefully remove the transfer membrane and place it in an Incubation Tray.
n. The rest of the contents of the cassette with the exception of the two external sponges can be discarded in the appropriate disposal now. Wash the sponges thoroughly with high purity water then air dry for next usage.
4. Blocking and Staining
a. Transfer the blot to an incubation tray containing ~25mL of high purity water after the transfer is complete and incubate for 5 minutes with rocking.
d. While the blot is being blocked, prepare the primary antibody solution. Transfer 10mL of Azure Chemi Blot Blocking Buffer to a 15mL conical tube then add primary antibody. Typical primary antibody dilutions range from 1:1000–1:5000.
e. After blocking, discard the Blocking Buffer in the container, while being very careful not to pour the transfer membrane out as well.
g. Wash the blot three times.
h. During the washing step, prepare a 1:50,000 dilution of secondary antibody solution. Transfer 10mL of Azure Chemi Blot Blocking Buffer to a 15mL Falcon tube then add 2μL of 1:10 diluted secondary antibody-HRP conjugate at 1 mg/mL. (To 18μL of Azure Chemi Blot Blocking Buffer add 2μL of stock antibody conjugate)
i. After the third wash, decant the washing solution and apply the diluted secondary antibody-HRP conjugate.
5. Application of Chemiluminescent Substrate
Note: Only begin this step if imaging preparations are complete.
a. Prepare the Azure Radiance Q (or Radiance Plus if higher sensitivity is required) reagent by mixing equal amounts of Radiance and Radiance Peroxide. (For one membrane combine 3mL of Reagent A with 3mL of Reagent B in a 15mL Falcon tube.)
b. Drain excess wash buffer from the blot. Then place it on a clean and dry surface, such as a blot development folder. Pour the working solution of substrate onto the membrane then allow it to incubate for 5 minutes.
c. Drain excess substrate from the blot. Place the blot in a development folder prior to imaging to keep it wet. Do not allow the membrane to dry as the HRP enzyme will no longer be active once the blot has dried.
Note: The Azure 600 Imaging System is referenced in this protocol, but any Azure Imager model that is equal to or greater than the Azure 280 Imaging System will work with this workflow for chemiluminescence.
a. (Optional, but recommended) Prior to imaging, place the treated membrane on a background Quenching Sheet to reduce background signal.
c. Turn on the Azure 600 by using the power switch on the back-right, then the green power button on the front.
e. Select the “Chemi Blot” option, choose between a system-calculated auto exposure with “Auto Image” or input specific desired settings in “Manually Image.”
f. After the image is taken, it will automatically appear in the Gallery tab. From here, contrast settings can be altered in “Adjustments,” the image can be saved in multiple image formats to the imager’s D drive or an external drive, as well as other options detailed in the Azure Imaging System User Manual.
Chemiluminescent detection excels at identifying small amounts of proteins of interest with high sensitivity. Images can then be analyzed through Azure Biosystems’ state-of-the-art analysis software AzureSpot Pro or other image analysis programs.
AzureSpot Pro combines several powerful analysis tools into one convenient and easy to use package. Designed to guide you through the analysis process, it is an easy-to-use Western blot image analysis software that makes complex and customized analysis simple. Try out AzureSpot Pro free by downloading a free trial.
Chemiluminescent Western blotting is a well-established technique that allows for the sensitive identification of a protein of interest transferred to a membrane. Whereas past laboratories relied on the tedious process of film development, modern imagers like the Azure 600 Imaging System allow for more simple, intuitive, and space-efficient visualization of chemiluminescent blots.
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