Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

1. Cell Culture, Stimulation, and Lysis

Note: This method can be used with many cell types. To illustrate the method, an example using human umbilical vein endothelial cells (HUVECs) is described.

1. Culture HUVECs on gelatin-coated, 10 cm Petri dishes in endothelial cell basal medium with appropriate supplementation (see Table of Materials), and also containing 5% FCS, epidermal growth factor (5 ng/mL), vascular endothelial growth factor (VEGF: 0.5 ng/mL), basic FGF (10 ng/mL), insulin-like growth factor (20 ng/mL), hydrocortisone (0.2 µg/mL), and ascorbic acid (1 µg/mL).
2. Starve cells overnight with endothelial cell basal medium supplemented with 1% FCS and no growth factor supplement.
3. Aspirate all medium, add 2 mL of endothelial cell culture medium without growth factors (starvation medium) in one dish (control), then add 50 ng/mL VEGF in 2 mL of starvation culture medium in a second dish for 7 min.
4. Aspirate the medium and wash cells 2 times with 10 mL room temperature PBS.
5. Ensure the Petri dishes are on ice and keep them there from this step onwards.
6. Add 250-400 µL of ice cold lysis buffer (Bicine/CHAPS; see Table of Materials) containing aqueous protease inhibitor mix and DMSO inhibitor mix (Phosphatase inhibitors; see Table of Materials) to each 10 cm plate. Swirl the plate to ensure proper coverage and keep it for 10 min on ice.
   NOTE: The final concentration of protease and the DMSO inhibitor mix should be 1x.
7. Scrape the cells from the dish with a cell scraper and transfer to a pre-chilled microfuge tube. Pipette up and down 5 times to lyse cells.
8. Briefly sonicate the cells at 4 °C. Set the sonicator as follows: number of cycles = 5, power = LOW, ON = 5 sec, OFF = 30 s. This step is included to break nucleic acids, and not to lyse cells.
   NOTE: Sonication should be mild to prevent denaturation of proteins.
9. Vortex the tube for 5 s (not continuously), 2 s at a time (3 times), and keep on ice.
10. Clarify lysate by centrifugation at 14,000 x g for 15 min at 4 °C, then immediately transfer the supernatant to a clean pre-chilled microfuge tube.
11. Measure the protein concentration using a Bicinchoninic Acid (BCA) protein assay kit⁴.
12. Make 10 µL aliquots, snap freeze in dry ice or liquid nitrogen, and store at -80 °C until use.

2. Sample Mix Preparation (Calculation for 150 µL Sample Mix)

1. First, prepare a sample diluent mix by adding 1 µL of DMSO inhibitor mix (stock 50x) and 2 µL of protease inhibitors (stock 25x) to 47 µL of sample diluent (see Table of Materials), so that the final concentration of DMSO inhibitors and protease inhibitors becomes 1x. Next, dilute the protein lysates using the sample diluent mix to obtain the desired concentrations (see step 2.3).
2. Prepare ampholyte/ladder/protease inhibitor/DMSO inhibitor mix by adding 3.325 µL standard ladder (see Table of Materials; stock 60x), 6 µL of protease inhibitor (25x), 3 µL of DMSO (50x) inhibitor to 137.675 µL ampholyte premix (see Table of Materials). Vortex the tube at least 15 s total, 5 s at a time (3-4 times), and keep on ice.
3. Mix the solutions from steps 2.1 and 2.2 in a 1:3 ratio, so that the final concentrations of DMSO, protease inhibitors, and the pI standard ladder become 1X, and the proteins in the capillary reach the final desired concentrations (e.g., 50 µg/mL was used for Figure 2).
   NOTE: Protein concentration in the capillary and well are the same.
4. Load 10 µL of sample mix into the appropriate well, according to the 384-well plate assay template layout (see step 3 and Figure 1) and keep the plate on ice.
5. Dilute the primary (pERK1/2, 1:50; ERK1/2, 1:100; HSP 70, 1:500) and secondary antibodies (1:300) with antibody diluent (see Table of Materials) and pipette 10 µL of primary and 15 µL of secondary antibodies into each well.
   NOTE: In general, higher concentration of antibodies are required for cIEF assays as compared to conventional western blots.
6. Mix Luminol and peroxide XDR (1:1 ratio; see Table of Materials) together and pipette 15 µL into each well.
   NOTE: Mix these solutions fresh every time before use and keep on ice.
7. Once samples and all reagents are pipetted into the plate, centrifuge the plate at 2,500 x g for 10 min at 4 °C to spin down the liquid and remove the bubbles. If bubbles still exist, remove them manually using a thin pipette tip.
   NOTE: Do not load more than 20 µL of sample or reagents per well; otherwise, the instrument pipette cannot be washed properly. Make sure to follow the instruction manual from the manufacturer to prepare all reagents.

3. Designing a New Assay Template with System Software (Figure 1)

1. Open the system software and click "new" from the File menu.
2. Go to the layout pane and assign locations for reagents and samples in a 384 well-plate. Use up to a maximum of 96 wells per 384-well plate.
3. Make a reagent assignment by clicking a well anywhere in the block.Select a row block of 12 wells each. Wells, e.g., from 1-12 or 13-24, are assigned as a block by default.
4. Go to the layout pane tool bar and insert either an empty row block or a sample, primary, secondary, or luminol row block. Each block is assigned a different color, so they are easy to differentiate.
   NOTE: When clicking a well, a black border can be seen around the block where the new block will be inserted in the pane. A row block can also be deleted.
5. Go to the protocol pane and select a reagent location in the plate. Then, click a cell in the sample column and select the reagent from the dropdown menu.
6. In this same fashion, select the reagent locations for the primary, secondary, and luminol for each cycle.
7. Click the cells, one at a time, in the primary or secondary antibody column, and change the incubation times, if necessary.
   NOTE: Entry notes for each cycle can also be added.
8. Add cycles by clicking the 'add' button in the protocol section. 1 cycle, 4 cycles, or all cycles (a maximum of 8 cycles can be added to the protocol).
   NOTE: It is possible to copy and paste cycle information in this step: select a cycle, go to edit, and copy and paste.
9. Enter information pertaining to the sample, such as the identity of the primary and secondary antibodies, their catalog numbers and dilutions, etc., in the template pane. This is an optional step that is useful during the post-run analysis.
10. Save the new assay file. Before clicking the 'start' button, quickly check the layout to make sure that everything is correct.

4. Protocol for cIEF Instrument Setting

1. Set capillary isoelectric focusing electrophoresis separation conditions. These should be 15,000 µW and 40 min, and the UV immobilization time should be 80 s. However, the UV immobilization time can be set within the range of 80-140 s, and may need to be optimized for the protein of interest.
   NOTE: A time point for UV immobilization can be set for each cycle, but not for each capillary.
2. Set wash 1 to 2 washes, for 150 s each wash (default), and primary antibody incubation for 120 min.
3. Set wash 2 to 2 washes, for 150 s each wash (default), and secondary antibody incubation for 60 min.
4. Set wash 3, which should be 2 washes, for 150 s each wash (default).
5. Set exposure times when chemiluminescence will be detected; these should be 30, 60, 120, 240, 490, and 960 s. All steps (1-8) will be carried out in a single capillary.

5. Running the Assay File

1. Once everything is ready, click start to begin the run. The software will prompt you to remove the waste, to refill the water and the capillary box, to add anolyte and catholyte to the resource tray, to add wash buffer and finally to load the plate into the cooled sample tray.
   NOTE: Remove the lid from the capillary box, but do not remove the lid from the assay plate. The lid from the plate can be removed automatically by the instrument when necessary. This helps to prevent reagent evaporation.
2. A couple of minutes after the run has been started, an instrument status bar will be displayed on the computer screen. Status messages and progress bars can be seen corresponding to the instrument's current stage.
   NOTE: Initially, the instrument will check if everything is correct before proceeding to pipette the anolyte and catholyte into the separation tray, picking the first block of 12 capillaries, removing the lid of the plate, and placing the samples from the sample plate into the capillaries, and then finally into the separation chamber for electrophoresis.
3. Click on the run summary screen to see two panes: status and separation. Users can view the run progress, and movies of the separation (the 12 capillaries of each cycle) can be stored when the electrophoresis step of each cycle is completed. To observe the electrophoresis separation in a capillary, play the separation movie for that capillary by clicking the desired cycle and then the play button in the control panel.
4. The run file can be stored automatically upon the completion of the run.

6. Analyze the Data (Figure S1)

1. Open the run file and select the analysis screen tab. In Figure S1A, the data shown (peaks) in the sample graph is from the selected capillary (i.e., 4^th capillary from cycle 1).
2. Click edit and then analysis (Figure S1B). At this stage, users can change the pI range, apply the appropriate pI standard for a given experiment, add a peak fit, and add a peak name.
   NOTE: When using a pH 5-8 ampholyte premix (as is the case here), the recommended standard ladder to use is one with fluorescently labeled peptides with pIs of 4.9, 6.0, 6.4, 7.0, and 7.3, and when using a pH 3-10 premix, the recommended ladder is one with pIs of 4.0, 4.9, 6.0, 6.4, and 7.3.²
3. Results are displayed in the Peaks tab; the values displayed for the samples are Position, pI, peak Height and Area, % Area, peak Width, and signal to noise (S/N) (Figure S1C). Choose which data to use, and export the data for further analysis.
   NOTE: Data can only be exported after labeling the peak (Figure S1C).