Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Podsumowanie

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

Streszczenie

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

Wprowadzenie

A western blot (also known as an immunoblot) is a powerful and fundamental technique across a broad range of scientific and clinical disciplines. This technique is used to examine the presence, relative abundance, relative molecular mass, and post-translational modifications of proteins¹. Combined with digital image analysis, this method can reliably analyze the abundance of proteins and protein modifications². Although western blot is performed routinely, it is a time-consuming and labor intensive method. Long incubations of the antibody with membranes are required. Here, we describe a modification of the incubation method that overcomes this limitation without sacrificing sensitivity.

During incubation of the membrane, antibodies float in solution while antigens are immobilized on the membrane. Because of their high affinity, the rate of antibody binding to the antigen is faster than the diffusion of antibodies from the bulk solution to the membrane. This creates a low concentration “depletion layer” (Figure 1). It may take hours for more distant antibodies to reach the membrane via passive diffusion, which is the main factor responsible for long incubation times in this technique. This effect is called the mass transport limitation (MTL)³. It has been proposed that repetitive draining and replenishing the antibody-containing solution may disrupt the depletion layer and overcome MTL⁴. Here, we have devised a unique technique that implements this cyclic draining and replenishing (CDR) concept to diminish the effect of MTL in a traditional western blotting protocol and remarkedly shorten the required incubation period.

In order to maintain the detection sensitivity with a short incubation time, we made use of an immunoreaction enhancing technology that accelerates the antigen-antibody reaction, thereby improving the signal-to-noise ratio⁵. Many commercially available immunoreaction enhancing agents (IRE) consist of 2 components (Solution 1 and 2, see Table of Materials). The proprietary composition or mechanism of action of IRE has not been disclosed, but we have previously found that IRE decreased the dissociation constant between the antigen and antibody in solid phase binding assays, indicating increased affinity is, at least in part, responsible for the enhancing effect of IRE⁶. The combination of CDR with IRE yields an ultra-high-speed western blotting protocol that reduces the entire procedure time without sacrificing sensitivity.

Protokół

1. SDS-PAGE and transfer to a PVDF membrane

1. Perform SDS-PAGE to separate proteins based on relative size.
   NOTE: Any commercial or home-made gel system is fine. Please follow the manufacturer’s protocol.
2. Transfer separated proteins from a gel onto a PVDF membrane.
   NOTE: Common transfer methods (semi-dry or wet transfer) are suitable. Please follow the manufacturer’s protocol.

2. Blocking of PVDF membranes

1. Incubate the membranes in appropriate blocking buffers for 1 h with agitation at room temperature.
   NOTE: Depending on the primary antibody and detection system, an appropriate blocking buffer should be selected. For example, the most typical blockers are BSA, nonfat dry milk, casein, and commercial synthetic polymers. PBS and/or TBS with 0.1% Tween20 are the most commonly used buffers. This blocking step may be done overnight at 4 ˚C.

3. Incubation with primary antibody

1. Prepare 10% immunoreaction enhancing agent-1 solution (IRE-1) with distilled water. Vortex well.
2. Prepare the diluted primary antibody with 10% IRE-1. Mix it gently and thoroughly.
3. If using a larger membrane (4 cm x 8 cm – 8 cm x 8 cm), add 8 mL of the antibody solution into 50 mL conical centrifuge tubes. If using a smaller membrane (2 cm x 8 cm – 4 cm x 8 cm), add 3 mL of the antibody solution into 14 mL round-bottom polypropylene tube (Table of Materials).
4. Pick up the PVDF membrane with tweezers and drain the blocking solution briefly. Insert the PVDF membrane into this tube with gloved fingers and ensure that the entire membrane adheres to the wall of the tube. When the PVDF membrane is inserted into a tube, face the “protein side” of the membrane that was originally in contact with the gel inward. Close the cap tightly.
5. Insert the 50 mL tube into a glass bottle for the hybridization oven.
   NOTE: When 14 mL tubes are used for this incubation, insert a 14 mL tube into the 50 mL tube, using a pair of plastic ring holders to keep the inner tube in the center of the 50 mL tube.
6. Turn on the hybridization oven.
7. Incubate the membrane with 6 rpm rotation for at least 5 min.
   NOTE: Make sure that the membrane adheres to the wall at all times and that the (inner) tube is horizontal to cover the membrane with the antibody solution evenly. Calibrate the dilution of the antibody and incubation time prior to the actual experiment.

4. Membrane wash

1. Stop the rotation.
2. Carefully remove the PVDF membrane from the tube with tweezers, and place the membrane into a container with 50 mL of PBS-T.
3. Rinse the membrane briefly with PBS-T.
4. Rinse the membrane in the container with distilled water until no bubbles appear. This is to remove the majority of the antibody.
5. Transfer the PVDF membrane from the container to the salad spinner that contains 250 mL of PBS-T.
6. Place the strainer basket in the spinner. Ensure that the lid has been put on securely.
7. Activate the spinner and run it for about 20-30 s.
8. Discard the solution and briefly rinse the inside of the spinner with distilled water.
9. Add 250 mL of PBS-T into the spinner again.
10. Repeat steps 4.6 and 4.7.

5. Incubation with the secondary antibody

1. Prepare 10% immunoreaction enhancing agent-2 solution (IRE-2) with distilled water. Vortex well.
2. Prepare the diluted secondary antibody with 10% IRE-2. Mix it gently and thoroughly.
3. Select the volume of the antibody solution and tube as stated in step 3.3.
4. Pick up the PVDF membrane with tweezers and drain the solution briefly. Insert the PVDF membrane into the tube as stated in step 3.4.
5. Insert the 50 mL tube into a glass bottle for the hybridization oven.
6. Turn on the hybridization oven.
7. Incubate the membrane with 6 rpm rotation for at least 5 min. Make sure that the membrane adheres to the wall at all times and that the (inner) tube is horizontal to evenly cover the membrane with the antibody solution.
   NOTE: Calibrate the incubation time prior to the actual experiment. When the secondary antibody is fluorescently conjugated, perform the incubation in the dark.

6. Membrane wash

1. Follow steps 4.1 to 4.10.

7. Image acquisition

1. Chemiluminescent detection
   1. Place a piece of semi-transplant flexible film (10 cm x 15 cm) on a flat surface.
   2. Mix 2 components of the chemiluminescent substrate in a 5 mL tube.
   3. Immediately transfer 1.5 mL of the mixed substrate to the semi-transplant flexible film and place the PVDF membrane upon it. Then, transfer the rest of the substrate solution onto the membrane.
   4. Incubate the PVDF membrane with the mixed substrate on a semi-transparent flexible film for 1 min.
   5. Pick up the PVDF membrane with tweezers and drain the substrate solution briefly.
   6. Sandwich the membrane between a pair of transparency films.
   7. Acquire the image under chemiluminescent mode.
2. Fluorescent detection
   1. Pick up the PVDF membrane with tweezers and drain the solution briefly.
   2. Sandwich the membrane between a pair of transparency films.
   3. Acquire the image under fluorescent mode.
      NOTE: Steps 7.1 and 7.2 should be performed in close proximity to the imaging machine to ensure maximum sensitivity.
   4. When the background signal is high, perform a washing step in a container with 80-100 mL of PBS-T: 10 min rinse 3 times for the primary antibody and 5 min rinse 6 times for the secondary antibody.
      NOTE: Both primary and secondary antibodies diluted with 10% IRE solutions can be used up to 8-10 times without loss of sensitivity⁶. The solution should be kept at 4 °C for up to 1 month.

Wyniki

This example illustrates the effectiveness of the CDR method together with immunoenhancing technology on western blot. Static incubation was performed on a semi-transparent flexible film where PVDF membranes were incubated with the antibody solution, whereas CDR incubation was in a hybridization oven by rotating tubes that contained the membranes and the antibody solution (Movie). Figure 2 showed quantities of antigen and antibody, respectively, necessary for chemiluminescent detection on we...

Dyskusje

Western blot is a widely used analytical technique to detect specific proteins that was developed ~40 years ago^7,8. Since then, the technique has continued to evolve, with subsequent innovations improving the sensitivity, speed, and quantitation of the technique^{2,9,10,11,12,}

Materiały

Name	Company	Catalog Number	Comments
14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap	Falcon	352059
Apollo Transparency Film for Laser Printers	N/A	N/A	Any kinds of Laser Printer Transparency Film are fine.
Azure Imaging System 600	Azure Biosystems	Azure 600	Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.
Can Get Signal Immunoreaction Enhancer Solution	TOYOBO	NKB-101
CARNATION Instant Nonfat Dry Milk	Carnation	N/A
ECL anti-mouse IgG, Horseradish peroxidase linked F(ab’)2 fragment from sheep	GE Healthcare	NA9310V
ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab’)2 fragment from donkey	GE Healthcare	NA9340V
HYBAID Micro-4	N/A	N/A	Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.
Immobilon-P PVDF Membrane, 0.45 µm pore size	Millipore Sigma	IPVH304F0
IRDye 680RD Goat anti-Mouse IgG Secondary Antibody	LICOR	926-68070
IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody	LICOR	926-32211
KPL LumiGLO Reserve Chemiluminescent Substrate	seracare	5430-0049
Mouse anti-ß actin antibody	Millipore Sigma	A5316
Odyssey Blocking Buffer (PBS)	LICOR	927-40100	Blocking Buffer for fluorescent detection
OXO Salad Spinner	OXO	32480V2B	Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.
Parafilm Sealing Film	Bemis	Parafilm M PM996	semi-transparent flexible film
Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes	Falcon	352070
Rings to hold 14 ml tube in the center of 50 ml tube	N/A	N/A	Prepared in a machine shop
Rabbit anti-6X His tag antibody	Abcam	ab9108
Tween20	Millipore Sigma	P2287