Immunoblot Analysis

Immuno blotting, often referred to as Western blotting is used to identify within a protein sample, specific antigens recognized by polyclonal or monoclonal antibodies. This procedure typically follows one or 2D gel electrophoresis and involves the transfer of separated proteins to nitrocellulose membranes. Incubation of these membranes with primary and secondary antibodies, directly or indirectly conjugated to enzymes and visualization of protein antigens on these membranes using colored or fluorescent substrate reactions.

In this video, we provide a step-by-step demonstration of an immuno blotting procedure. Hi, I'm Sean Gallagher from UVPI collaborate with a proteomic center here at the KE Graduate Institute. Today I'm gonna be showing you immuno blotting analysis.

It involves a number of steps including protein separation, blotting of the proteins onto membranes, immuno probing and visualization with chromogenic and kim luminescence substrates. So let's get started. Before starting the immuno blotting procedure, the protein samples must first be separated by electrophoresis using small or standard sized one or two dimensional gels.

Prepare the antigenic samples and load them into the lanes on the gel, include prestained or biotinylated protein, molecular weight standards in one or more of the gel lanes. These protein markers will transfer to the membrane and conveniently indicate membrane orientation and sizes of proteins after immuno staining. Once the electrophoresis is complete, we are ready to assemble the immuno blot.

To assemble the immuno blot First, disassemble the gel sandwich and remove stacking gel equilibrate the gel for 30 minutes At room temperature in transfer buffer, this equilibration is required to prevent a change in the size of the gel during transfer while the gel is equilibrating. Begin assembling the transfer sandwich in a tray large enough to hold the plastic transfer cassette fill with transfer buffer so that the cassette is covered. Now place a scotch bright pad or sponge on the bottom half of the plastic transfer cassette.

Then take a sheet of filter paper that has been cut to the same size as the gel. Pre-wet it with transfer buffer and place it on top of the scotch bright pad. Once the 30 minute gel equilibration is done, place the gel on top of the filter paper.

Remove any air bubbles between the gel and filter paper by gently rolling a test tube or glass rod over the surface of the gel. Or you may use the BioRad roller, which comes with the BioRad kit. Remember to use gloves when manipulating filter papers, gels and membranes.

Oil from your hands will block the transfer. Next, prepare the transfer membrane. Cut the membrane to the same size as the gel plus one to two millimeters on each edge.

Then place the membrane into distilled water slowly with one edge at a 45 degree angle, the water will wick up into the membrane and eventually wet the entire surface. If it is inserted two quickly into the water, air gets trapped and will appear as white blotches in the membrane. The protein will not transfer onto these areas.

Once the membrane is completely wet, equilibrate it for 10 to 15 minutes and transfer buffer. This wetting procedure works for nitrocellulose and nylon membranes. Only PVDF membranes are hydrophobic and will not wet simply from being placed into distilled water or transfer.Buffer.

PVDF membranes must first be immersed in 100%methanol for one to two seconds, then equilibrate for 10 to 15 minutes. In transfer buffer. Do not let the membrane dry out at any time.

If this occurs wet once again with methanol and then the transfer buffer. Once the membrane is ready, place the pre wetted membrane directly onto the top side of the gel. Remember to remove all air bubbles between the gel and the membrane by gently rolling a test tube glass rod or BioRad roller over the surface of the membrane.

Next wet, another piece of watman three mm filter paper. Then place it on top of the membrane and again, remove all air bubbles. Then place another scotch bright pad or sponge on top of this filter paper.

Complete the assembly of the immuno blot sandwich by locking the top half of the transfer cassette into place. Now that the immuno blot sandwich is assembled, we are ready to transfer the proteins from the gel to the membrane. To begin the transfer procedure, fill the electro blotting tank with transfer buffer and place the transfer cassette containing the sandwich into the electro blotting apparatus.

It is important to the sandwich so that the membrane faces the anode or positively charged side of the tank. Connect the leads of the power supply to the corresponding anode and cathode sides of the electro blotting apparatus. This particular criterion blotter comes with a cooling ice block.

Now, electrophoretic transfer proteins from gel to membrane for 30 to 60 minutes at 50 volts with cooling or overnight at 14 volts in a cold room. When the transfer is done, turn off the power supply and disassemble the apparatus. Then remove the membrane from the blotting apparatus and note the orientation by cutting a corner.

At this point, membranes can be dried and stored in a resealable plastic bag at four degrees Celsius for one year. Prior to further processing, dried PVDF membranes must be placed into a small amount of 100%methanol to wet the membrane. Then in distilled water to remove the methanol.

Once the orientation is marked, stain the gel to verify transfer efficiency. To visualize transferred proteins, you may either stain the membrane reversibly with cipro ruby or irreversibly with kamasi blue India ink, naphthol blue, or colloidal gold. These irreversible staining procedures are incompatible with nylon membranes.

Now that the proteins are transferred to the membrane, proceed with immuno probing and visual detection of the proteins. In this step, immobilized proteins on the membrane are probed with specific antibodies to identify and quantitate any antigens present. To begin place the membrane in a plastic incubation tray with 20 milliliters blocking buffer.

Some people use bags, but the trays are often used instead as they are especially useful when processing large numbers of strips in different primary antibody solutions. Next, incubate the sealed membrane for 30 to 60 minutes At room temperature with agitation on an orbital shaker or rocking platform. While the membrane is incubating dilute the primary antibody and blocking buffer.

The dilution is determined empirically, but is typically one to 100 to one to 1000. For a polyclonal antibody, one to 10 to one in 100 for hybrid supernatants and greater than one to 1000 for murine acids fluid containing monoclonal antibodies. When the incubation is done, pour out the blocking buffer.

Replace the buffer with diluted primary antibody, and again, incubate for 30 to 60 minutes at room temperature with constant agitation. Next, wash the membrane four times by agitating with 100 milliliters. TTBS for NITROCELLULOSE or PVDF filters or TBS for nylon filters.

Wash for 10 to 15 minutes each time while membrane is being washed, dilute the secondary antibody in blocking buffer. Examples of secondary antibodies include horse radish peroxidase or alkaline phosphatase, anti IG conjugate, commercially available enzyme conjugated. Secondary antibodies are usually diluted one to 200 to one to 25, 000 prior to use.

After the wash steps are done and the wash materials discarded, add diluted horse radish peroxidase or alkaline phosphatase, anti IG conjugate and incubate 30 to 60 minutes at room temperature with constant agitation. After 30 to 60 minutes, remove membrane from the bin and wash it four times, 10 to 15 minutes each time. In TTBS as described earlier.

Once the washes are complete, we are ready to proceed with the visualization step. But first, we will demonstrate an alternative immuno probing procedure. This alternative immuno probing procedure is based on the vector stain A BC kit from Vector Labs.

It uses an avidan biotin complex to attach horse radish peroxidase or HRPO or alkaline phosphatase A A P to the biotinylated secondary antibody. Today we will be demonstrating the HRPO kit. TTBS is well suited as a blocking buffer for avadon biotin systems.

But for nylon filters, protein binding reagents are recommended because non-fat dry milk contains residual biotin, which will interfere with the immunoassay. It must be used in the blocking step only to begin equilibrate the membrane in blocking buffer in an open tray with constant agitation using an orbital shaker or rocking platform. Incubate the membrane for 30 to 60 minutes at room temperature while the membrane is equilibrating.

Prepare the primary antibody solution. Use TTBS for NITROCELLULOSE or PVDF membranes with high sensitivity Aden. Biotin systems.

Dilution of cera containing primary antibody generally range from one in 1000 to one in 100, 000. The use of chemiluminescent chemistry requires the higher range of dilutions. In our case, demonstrating chromogenic.

We are using a one in 5, 000 dilution of the primary. Once the e equilibration is done, remove the blocking buffer. Then add enough primary antibody solution to cover the membrane.

Incubate the membrane for 30 minutes at room temperature with gentle rocking after 30 minutes. Wash the membrane three times in TTBS for nitrocellulose at five minute intervals During the washing step, prepare the biotinylated secondary antibody solution by diluting one drop of biotinylated antibody with 10 milliliters of TTBS. Once the washing steps are done, add the secondary antibody solution.

Incubate the membrane for 30 minutes at room temperature with slow rocking while the membrane is being incubated with secondary antibody. Prepare the avadon biotin HRPO to do this. Mix two drops of vti stain reagent A and two drops reagent B into 10 milliliter.

TTBS for nitrocellulose. Incubate for 30 minutes at room temperature when the secondary antibody incubation is complete. Wash membrane three times over a 15 minute span with TTBS or TBS.

Next, add the avidan biotin enzyme solution to the membrane and incubate for 30 minutes at room temperature with slow rocking. Then wash the membrane three times at 10 minute intervals with TTBS. Now that the immuno probing procedure is complete, the membrane bound proteins are ready to be visualized with chromogenic substrates.

For this final visualization step, the substrates four CN dab slash ICL two and TMB are commonly used with horse radish, peroxidase based immuno detection procedures. If the final membrane wash during the immuno probe procedure was performed in TTBS then washed the membrane for 15 minutes at room temperature and 50 milliliters. TBS now place the membrane into chromogenic visualization solution.

Protein bands should appear in 10 to 30 minutes. After the 30 minute incubation, discard the substrate reaction mixture and terminate the reaction by adding distilled water. Immuno blotting is a multi-step procedure used by thousands of labs to detect the presence of specific proteins following one or 2D electrophoresis.

I've just shown you how to perform an immuno blood analysis. So that's it. Good luck with your experiments and thanks for watching.