A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The goal of this procedure is to detect and accurately quantify the relative abundance of specific proteins within a complex biological sample using quantitative, fluorescent twist and blotting. This is accomplished by first electro fluorescently separat protein extracts using a precast gradient gel. In the second step, the equivalent protein load is verified across all sample lanes through the application of a total protein stain, and then the eye block semi-dry fast transfer system is used to transfer the samples onto A-P-V-D-F membrane.

Finally, the proteins are probed with specific primary antibodies followed by the appropriate fluorescent tagged secondary antibody to facilitate their sensitive, robust and linear detection at infrared and far red wavelengths. Ultimately, quantitative fluorescent western blossoming can be used to reproducibly detect, visualize, and accurately quantify a large number of membrane bound and soluble proteins across a broad range of tissue samples. The main advantage of this technique over existing methods such as ECL based imaging, is that it allows a greater sensitivity with enhanced precision on a broad linear scale for a truly quantitative readout Begin by manually macerating the tissue sample, followed by homogenization in freshly prepared extraction buffer at approximately one to 10 tissue weight per buffer volume until a smooth consistent homogenate is produced.

Next, centrifuge the cell lysate for 20 minutes at 20, 000 times G at four degrees Celsius. Then transfer the sate containing the solubilized proteins into a clean micro centrifuge tube and store both the pellet and S supernatant fractions at minus 80 degrees Celsius until required. After determining the protein concentration of the supinate, transfer 15 micrograms of protein per sample into new micro centrifuge tubes.

Then bring the final volume of each sample up to 10 microliters with distilled water and add five microliters of Forex loading buffer. Thoroughly agitate the cell solutions by vortexing, and then heat the samples at 98 degrees Celsius for two minutes to denature the proteins. After denaturation, add three microliters of an appropriate molecular weight standard into the first well of each of the experimental and loading control gels.

Thereafter, load 10 microliters of each sample into the appropriate subsequent wells following electrophoresis. Use a gel knife to release the gels from the cassette and remove the wells and the foot of the gel. Mark the gel to aid in later orientation.

Then to determine the equivalent protein load decant approximately 30 milliliters of protein stain into a square 12 by 12 centimeter Petri dish and place the loading control gel into the stain, ensuring that the solution covers the entire gel. Gently agitate the gel for one hour minimum at room temperature, and then discard the protein stain and wash the gel three times in distilled water prior to visualization for the eye block. Two semi-dry fast protein transfer Following electrophoresis, prepare the bottom transfer stack by removing the foil cover and pre wetting the membrane with running buffer directly from the gel tank.

Rewet the filter paper with distilled water at this time as well. Next, lace the experimental gel onto the prepared bottom transfer stack, and then lay the pre-work filter paper over the gel and roll them both to ensure that no bubbles are trapped between the layers. Lay the top stack on top of the filter paper and bottom stack.

Then roll the transfer stack sandwich to eliminate any air bubbles. Insert the sponge from the transfer stack kit onto the absorbent pad and place the transfer stack sandwich into the dedicated space. Then close the lid securely and start the I block two, transferring the proteins for seven minutes on program three.

When the program is finished, remove the transfer stack from the eye block machine and peel back the top transfer stack and filter paper layers exposing the gel on the bottom transfer stack. Use a scalpel to cut around the gel taking care to make a triangular cut for orientation purposes on the membrane following the membrane trimming to check the transfer efficiency protein stain, the gel as just demonstrated, and then swiftly moved the membrane into a clean 50 milliliter tube containing five milliliters of PBS for the first of three, five minute washes on a mechanical roller. After the third wash, discard the wash buffer and incubate the membrane in undiluted blocking buffer for a minimum of 30 minutes at room temperature.

Next, incubate the membrane in primary antibody diluted in five milliliters of blocking buffer and 0.1 between 20 overnight at four degrees Celsius with constant agitation. The next morning after discarding the primary antibody solution, wash the membrane six times for five minutes in PBS as just demonstrated. Then incubate the membrane in the dark with fluorescent tagged secondary antibody, dilated in five milliliters of blocking buffer and 0.1%between 20 with constant agitation, followed by six five minute PBS washes for all imaging.

First, log into the computer and imager. Then open the imaging software to image the western blot. First, open the lid of the imager and pour a small amount of PBS onto the glass.

On the bottom left hand corner of the imager, place the membrane on top of the PBS ensuring that it is square with the axis on the imager, and that a one centimeter gap is left between the membrane and the grid axis. Roll the membrane to ensure that no bubbles are trapped between the glass and the membrane, and then count the number of squares the membrane occupies on the x and y axis. Next, close the lid of the imager and enter the number of squares into the computer software.

Select the appropriate infrared channel and the appropriate intensity, and start the scan to visualize the total protein stain of the loading control gel. Repair the gel and the imager as just demonstrated for the experimental membrane, and then select the 700 channel. Set the intensity to five and start the scan.

To quantify the scanning data. Begin by adjusting the brightness and contrast buttons of the scanned file to produce the best image quality, rotating the images necessary to achieve the desired orientation. Next, select a rectangle from the shapes menu and use it to draw a rectangle around the entire lane.

In sample lane one. Copy and paste the shape around lane one to sample lane two. After confirming that the background measurement does not incorporate a signal into the next lane, draw a rectangle around each lane for the rest of the sample lanes.

Then display the signal for the shapes table for each rectangle drawn in arbitrary fluorescent units and save the image as a tiff bile. There are a number of control measures that are crucial for ensuring that accurate data is collected during quantitative fluorescent Western blotting. For example, it is desirable to include positive control samples.

Second, it is important to optimize the transfer time to guarantee an equivalent movement of high and low molecular weight proteins from the gel to the membrane. Third, the appropriate primary and secondary antibody dilutions must be determined to avoid the incorrect interpretation of the proteins of interest due to non-specific banding as illustrated in these images. Fourth, it is important to consider whether a secondary antibody raised in a different host species may be necessary, particularly in cases where an expected protein band is not detected.

Fifth, the production of a loading control gel can confirm the uniformity of the sample load. When combined with a total protein analysis, the gel can then be used to compare and quantify the protein load in each lane at various molecular weight ranges as measured against each sample. Finally, this technique lends itself to the stripping and re probing of membranes with more flexibility than kemi luminescence due to factors including but not limited to, an increased sensitivity, a reduced background, dual color detection, and membrane stability under long-term storage conditions.

After watching this video, you should have a good understanding of how to use quantitative fluorescent western plotting to detect and accurately quantify proteins of interest, as well as how to troubleshoot common problems that can occur when performing this technique.