This method can help answer key questions in the biomedical research field, such as biomarker discovery, drug discovery, diagnostics, and screening of drugs or antibodies. The main advantage of this technique is that it is high-throughput and can detect protein isoforms in a highly reproducible manner. The implications of this technique extend towards the diagnosis of many diseases because it can measure affected proteins or their isoforms.
Though this method can provide insight into cell-signaling pathways, it can be also be applied to other systems where specific proteins need to be analyzed. Visual demonstration of this method is critical as the assay steps are difficult to learn because of the tricks, such as removing bubbles, loading the plate, et cetera. First, prepare a sample diluent mix by adding one microliter of DMSO inhibitor mix and two microliters of protease inhibitors to 47 microliters of sample diluent, so that the final concentration of DMSO and protease inhibitors is one X.Dilute previously isolated protein lysates using the sample diluent mix to obtain the desired concentrations.
Next, add 3.325 microliters of standard ladder, six microliters of protease inhibitor, and three microliters of DMSO inhibitor to 137.675 microliters of antholite premix. Vortex the sample at least 15 seconds and keep on ice. Mix the two prepared solutions in a one to three ratio so that the final concentrations of DMSO protease inhibitors and the isoelectric point standard ladder are one X and the proteins in the capillary reach the final desired concentrations.
After the proteins are added to the sample mixture, all the steps will be performed on ice or at four degrees Celsius. Place a 384-well plate on ice. Load ten microliters of the sample mix into the appropriate well of the plate, according to the assay template layout designed with the automated Western blotting system software.
Following this, dilute the primary and secondary antibodies with antibody diluent. Then, pipette ten microliters of primary antibodies and 15 microliters of secondary antibodies into each well. Next, mix luminol and peroxide XDR in a one to one ratio.
Pipette 15 microliters of the luminol peroxide mix into each well. Once the samples and reagants have been added to the plate, centrifuge for ten minutes at 2500 times G and four degrees Celsius to spin down the liquid and remove the bubbles. If bubbles still exist, remove them manually using a thin pipette tip.
Open the automated Western blotting system software and click new'from the file menu. Go to the layout pane and assign locations for reagants and samples in the 384-well plate. Make a reagant assignment by clicking a well anywhere in the block.
Select a row block of 12 wells each. Next, go to the layout pane tool bar and insert a sample, primary, secondary or luminol row block. Go to the protocol pane and select a reagant location in the plate.
Then, click the cell in the sample column and select the reagant from the drop-down menu. In the same fashion, select the reagant locations for the primary, secondary, and luminol for each cycle. Click the cells one at a time in the primary or secondary antibody column and change the incubation times if necessary.
Then, add cycles by clicking the Add'button in the protocol section. In the template pane, enter information pertaining to the sample, such as the identity of the primary and secondary antibodies, their catalog numbers and dilutions. Save the new assay file.
Before clicking the start'button, quickly check the layout to make sure that everything is correct. Now, click start'to begin the run. The software will prompt the user to remove the waste, to refill the water and capillary box, to add anolite and catholite to the resource tray, to add wash buffer, and to load the plate into the cooled sample tray.
An instrument status bar will be displayed on the computer screen a couple of minutes after the run has started. Click on the run summary'screen to see the status and separation panes. To observe the electrophoresis separation in a capillary, play the separation movie for that capillary by clicking the desired cycle and then clicking the play button in the control panel.
Open the run file and select the analysis screen tab. Click edit'and then analysis'to change the isoelectric point range. Apply the appropriate isoelectric point standard.
Add a peak fit, and add a peak name. Finally, select the data to use in the peaks tab and export the data for further analysis. The electropherogram of phosphorylated extracellular signal-regulated kinases from vascular endothelial growth-factor stimulated lysates is shown here.
There's very high induction of phosphorylated proteins observed with vascular endothelial growth factor. The inset shows the endogenous loading control, HSP 70, indicating similar loading of samples for both untreated and treated samples. In a conventional immunoblot, only two bands were detected, even though the phosphorylated extracellular signal-regulated kinase proteins were resolved into four peaks by capillary isoelectric focusing analysis.
The electropherogram of extracellular signal-regulated kinases from vascular endothelial growth-factor stimulated lysates is shown here. The inset shows the same loading control used in parallel. A conventional immunoblot shows only two bands corresponding to extracellular signal-regulated kinase one and extracellular signal-regulated kinase two.
Whereas with the capillary isoelectric focusing, the extracellular signal-regulated kinase proteins were resolved into six peaks, corresponding to all four different phosphorylated peaks and two un-phosphorylated peaks. Once mastered, this technique can be done in a few hours, depending on the number of cycles, if it is performed properly. While attempting this procedure, it is important to remember to use higher antibody concentrations as compared to conventional immunoblots.
Visual demonstration of this method is critical as the assay steps are difficult to learn because of tricks, such as removing bubbles, loading the plate, et cetera. After watching this video, you should have a good understanding of how to design an assay, prepare samples, run the assay, and analyze the data to observe different isoforms of your protein.
