BioRad V three Workflow provides researchers with more reliable quantitation and confidence in the Western blotting process. This is accomplished by first using TGX stain free gels to separate and visualize protein samples. After separation proteins are transferred to a membrane using the blot turbo rapid transfer system, which allows verification of the transfer quality and efficiency.
After verifying transfer, the membrane is blocked and probed with primary and secondary antibodies for the target proteins. Finally, target protein levels are normalized using the stain free total protein measurement as a loading control. The V three workflow brings convenience and transparency to the western blotting process, providing researchers with confidence at every step from separation to transfer and quantitation.
The main advantage of the V three Western workflow over traditional Western blotting is that stain free technology offers a practical, convenient, and reliable total protein loading control for normalizing your target protein signals. This method can help answer key questions related to the reliability of loading control methodologies for the Western blotting technique that involve the oversaturation of housekeeping protein signals and the differential expression levels of housekeeping proteins under varying experimental conditions. We discovered that proteins activated in stain free gels could also be visualized on blos without further staining, allowing them to be used as an alternative to conventional total protein stains, as well as housekeeping protein loading controls.
In the next few minutes, we will demonstrate the complete V three workflow using multiplex fluorescence. The same procedure may also be used for chemiluminescence after preparing protein samples according to the text protocol. Using a criterion TGX stain free any KD precast gel, remove the comb and the tape from the bottom of the cassette.
Place the cassette in the gel apparatus and fill both reservoirs with running buffers. Load the protein standards and samples. Then run the gel for 20 minutes at 300 volts.
After electrophoresis is complete, use the gel cassette opening tool on the Criterion Cell lid to open the cassette. Then apply a few milliliters of water to the UV transluminator of the Chemi Dock MP imager. Carefully lift the gel from the cassette and place it on the UV Trans Illuminator Launch Image Lab software and set up a new single channel protocol for stain free gels using the default one minute gel activation time, select the appropriate gel type and default auto optimization for intense bands.
Then click run protocol. Once image capture is complete, visualize separation and sample quality. Then immediately proceed to the transfer step.
To carry out protein transfer, open a trans block turbo MIDI PVDF transfer pack. Place the bottom stack in the center of the cassette base. Remove the gel from the imager.
Align the gel on top of the membrane. Gently use the blot roller to remove air bubbles between the gel and the membrane. Next, align the top stack on top of the gel.
After removing any air bubbles, place the cassette lid on the base. Press the lid down firmly turn the dial clockwise to lock and insert the cassette into one of the two blotter bays. From the home screen, select the turbo protocol.
Choose the two mini or one mini gel, and press the run button for the appropriate bay. The transfer will be complete in seven minutes. Remove the cassette from the bay.
Unlock the cassette and disassemble the blotting sandwich. Place the post transfer gel on the UV transluminator of the Chemi dock MP imager, and immediately place the membrane in deionized water. Set up a new single channel protocol for stain free gels without an activation time.
Select the appropriate gel type and manually set the exposure time to that of the pre transfer gel and click run protocol. Once image capture is complete. Verify transfer efficiency by comparing sample band intensities between the pre and post transfer gels.
Remove the gel from the sample tray as it is no longer needed and discard. To verify, transfer to the blot. Place the membrane on the sample tray and set up a new single channel protocol.
For stain free blot. Select the appropriate gel type and default auto optimization for intense bands. Then click run protocol.
Once image capture is complete, verify transfer to the membrane. Remove the blot membrane from the UV trans illuminator and place it in blocking buffer for western blotting. After incubating the membrane at room temperature for one hour, incubate it with mouse and rabbit primary antibodies overnight at four degrees Celsius the following day.
Pour out the antibody solution and use 50 milliliters of TBST to wash the blot for five minutes. Incubate the blot with fluorescent conjugated, anti muse and anti rabbit secondary antibodies for one hour at room temperature. Then use TBST to wash the blot five times for five minutes each.
To image the blot, place it on the UV trans illuminator of the Chemi doc MP imager in image lab software. Set up a new multi-channel protocol. Configure channel one by selecting dite six 50 under blot applications and use the default auto optimization for intense bands.
Repeat these steps to configure channels two and three for DITE 5 49 and stain free blot respectively. Select the appropriate gel type and click run protocol. To define lanes, select the stain free blot image lane and bands.
Then automatic lane finder for accurate quantitation. Ensure that the background profiles in each sample lane are similar by adjusting the rolling disc size to 70. Confirm background uniformity by scanning through all lane profiles to define bands in both the dite six 50 and 5 49 channels.
Use the band finder tool to detect bands using the default sensitivity settings. To designate the normalization channel return to the analysis toolbox. Click normalization and choose stain free blot, ensuring that total lane protein is selected.
To assign the molecular weight standard lanes return to the analysis toolbox, click MW analysis tools and check the box under the appropriate lane. Finally, to view the normalized protein band intensities, click the analysis table In the toolbar. The software will automatically generate a table that contains volume intensities, normalization factors, and normalized volumes.
The normalized volumes should be used for data analysis and reporting. The table can be exported. For further analysis, we have demonstrated the complete V three Western blotting workflow.
Now we will show you representative results from a real experiment. Lysates of control and irradiated lymphoblasts cell cultures were analyzed using the V three Western blot workflow. The stain free total protein signal appears in blue.
The target protein M CM seven is shown in red, and the validated housekeeping protein loading control GA DH is shown in green. GA DH has been validated as a suitable loading control by assessing its linearity using the experimental conditions described in this video. The stain free total protein signals for each lane on the blot were measured using image lab software.
MCM seven signal intensities were normalized using both the total protein and GA DH signals. MCM seven protein band volumes normalized by both methods revealed 25%decreased expression levels in irradiated samples relative to control samples before accurate normalization. The housekeeping protein requires validation confirming its linear range and consistent expression levels among samples without validation.
Housekeeping protein loading controls do not guarantee accurate normalization. By contrast, total protein normalization does not require validation. Thus, the stain free total protein signal obtained by the V three workflow offers a practical, convenient, and more reliable loading control While attempting this procedure.
Remember that stain free technology permits fluorescent visualization of protein samples through a UV induced covalent modification of available tryptophan residues. Only in rare instances will this modification significantly impact downstream applications such as immuno detection or mass spectrometry. After watching this video, you should have a good understanding of how to perform the V three Western workflow and stain free total protein normalization.
Although we demonstrated this workflow for multiplex fluorescent Western blotting, stain free technology can also be used for total protein normalization with luminescent Western blots.
