Overview: How to fill the Simple Western Jess plate with ASSIST PLUS

The ASSIST PLUS pipetting robot and D-ONE single channel pipetting module, together with the Simple Western Jess, automate all the liquid handling steps required to analyze 24 samples, providing a complete walk-away solution for western blot protocol automation.

The whole workflow consists of 2 steps (Figure 1):

1. Simple Western Jess plate filling with D-ONE and ASSIST PLUS
2. Protein analysis with Simple Western

This application note provides Jess plate filling protocols for chemiluminescence detection of single targets, multiple target (using RePlex) and total protein analysis of prepared samples and reagents for reliable downstream protein analysis using Simple Western.

Downloads: App note and protocols for Jess plate set-up with the ASSIST PLUS pipetting robot

Plate set-up for RePlex chemiluminescence western blot protocol automation

Experimental set-up:

Position A: Wash buffer (blue)

Position B: B1 – antibody diluent (lavender), streptavidin-HRP (red), luminol-peroxide mix (yellow), RePlex mix (pink); B2 – primary antibody probe 1 (light green-1), secondary antibody probe 1 (green-1), primary antibody probe 2 (light green-2), secondary antibody probe 2 (green-2); B3 – biotinylated ladder (violet); B4-B6 – prepared samples (orange, arrow indicates processing direction)

Jess plate set-up for RePlex

Place a 25 ml reservoir on deck position A and fill it with at least 8 ml of wash buffer. Place the INTEGRA tube rack on position B with the specific reagent and sample tubes, as indicated in the experimental set-up. As shown in Figure 2, the Jess plate is in landscape orientation on position C. LLD enables the use of different aliquot sizes, and D-ONE informs the operator if the liquid volume is insufficient.

Equip ASSIST PLUS with the 0.5-300 µl D-ONE, and run the VIALAB program 'Jess_plate_setup_RePlex'. D-ONE and ASSIST PLUS with 12.5 µl long, sterile, filter GRIPTIPS transfers 10 µl of antibody diluent from a 2 ml tube in position B1 (Figure 2, lavender) to wells B1-B25, C1, E1 and F1 of the Jess plate (Figure 3) in position C. A 1 µl pre- and post-dispense guarantees precise pipetting while preventing bubble creation during dispensing. By automatically changing GRIPTIPS between different reagents or samples, D-ONE transfers 10 µl of primary antibody probe 1 from a 1.5 ml microcentrifuge tube in B2 (Figure 2, light green-1) to wells C2-C25 (Figure 3) of the Jess plate in position C (Figure 4a). From the second 2 ml screw cap vial in B1 (Figure 2, red), D-ONE transfers 10 µl of streptavidin-HRP/NIR to well D1 (Figure 3). Wells D2 to D25 (Figure 3) are filled with 10 µl secondary antibody probe 1 from a 1.5 ml microcentrifuge tube in B2 (Figure 2, green-1). 10 µl primary antibody probe 2 from position B2 (Figure 2 , light green-2) is transferred to wells E2 to E25 (Figure 3), and 10 µl secondary antibody probe 2 from B2 (Figure 2, green-2) is transferred to wells F2 to F25 (Figure 3).

With 300 µl sterile, filter GRIPTIPS, D-ONE transfers 500 µl of wash buffer (Figure 2, blue) from the 25 ml reservoir on position A to the Jess plate compartments in 2 steps (Figure 4b), as indicated in Figure 3. A slow speed (5) prevents bubble creation during buffer dispensing. Afterwards, 170 µl of luminol-peroxide mix is transferred from the 2 ml tube in B1 (Figure 5, yellow), and 300 µl of RePlex reagent mix from another 2 ml tube in B1 (Figure 5, pink) to the compartments indicated in Figure 3. D-ONE then transfers the RePlex reagent mix in 2 steps, with a pre- and post-dispense of 10 µl to prevent bubble creation during dispensing.

5 µl of biotinylated ladder is transferred from a 0.5 ml microcentrifuge tube in position B3 (Figure 2, violet) to well A1 (Figure 3). As indicated by the arrow in Figure 2, D-ONE transfers 3 µl of prepared sample (Figure 2, orange) from each 0.5 ml microcentrifuge tube in positions B4 to B6 (Figure 2, orange) to wells A2-A25 (Figure 3). Fast dispensing (speed 10) increases the accuracy for small volumes. The pipette then instructs the user to centrifuge the plate for 5 minutes at 2500 rpm, before proceeding with Simple Western Jess protein separation and immunodetection.

Tips:

• It is possible to work with very low volume inputs (5 µl) for precious samples, thanks to the D-ONE module's broad volume range.
• A prompt can be included in the protocol before starting with the sample transfer to allow the user to prepare the samples during reagent transfer.
• VIALAB's simplified programming allows plate set-up to be easily adjusted to perform western blot normalization by replacing the second chemiluminescence detection with a total protein analysis, as indicated in the kit insert.

Plate set-up for chemiluminescence western blot protocol automation

Experimental set-up:

Position A: Wash buffer (blue)

Position B: B1 – antibody diluent (lavender), streptavidin-HRP (red), luminol-peroxide mix (yellow); B2 – primary antibody (light green), secondary conjugate (green); B3 – biotinylated ladder (violet); B4-B6 – prepared samples (orange, arrow indicates processing direction)

Jess plate set-up for chemiluminescence western blot

Prepare a similar deck set-up to the RePlex western blot protocol, but without the RePlex reagent mix or the primary and secondary antibodies for probe 2 (Figure 5).

Select and run the VIALAB program 'Jess_plate_setup_chemiluminescence'. Following a similar procedure as for RePlex chemiluminescence detection, D-ONE starts by transferring 10 µl of antibody diluent (Figure 5, lavender) to wells B1-B25 and C1 of the Jess plate (Figure 6) in position C. 10 µl of primary antibody (Figure 5, light green) is then transferred to wells C2-C25, 10 µl of streptavidin-HRP/NIR (Figure 5, red) to well D1, 10 µl of secondary conjugate (Figure 5, green) to wells D2 to D25, and 15 µl of luminol-peroxide mix to all wells of row E, as indicated in Figure 6.

D-ONE follows with the transfer of the wash buffer (Figure 5, blue), biotinylated ladder (Figure 5, violet) and samples (Figure 5, orange), as described for RePlex chemiluminescence detection, and instructs the operator to centrifuge the plate before proceeding with the automated western blot.

Plate set-up for total protein western blot protocol automation

Experimental set-up:

Position A: Wash buffer (blue)

Position B: B1 – antibody diluent (lavender), total protein labeling reagent (light green), total protein streptavidin-HRP (green), luminol-peroxide mix (yellow); B3 – biotinylated ladder (violet); B4-B6 – prepared samples (orange, arrow indicates processing direction)

Jess plate set-up for total protein analysis

Set up the ASSIST PLUS deck in a similar way to the chemiluminescence detection protocol, but with the reagents for total protein analysis (Figure 7).

Select and run the VIALAB program 'Jess_plate_setup_total_protein'. D-ONE automatically selects GRIPTIPS, and transfers 10 µl of antibody diluent (Figure 7, lavender) into wells B1 and C1 to C25, 8 µl of total protein streptavidin-HRP (Figure 7, green) to row D, 15 µl of luminol-peroxide mix (Figure 7, yellow) to row E, and wash buffer from the reservoir to the compartment of the Jess plate, as indicated in Figure 8. Similar to the western blot protocol for chemiluminescence detection, 5 µl of biotinylated ladder (Figure 7, violet) and 3 µl of each sample (Figure 7, orange) are also transferred to the Jess plate in position C. The run is completed by the transfer of 10 µl of total protein labeling reagent (Figure 7, light green) into wells B2 to B25, as shown in Figure 8. After finishing the liquid transfers, the instrument instructs the operator to centrifuge the plate, as indicated in the kit.

Results

Western blotting can be very time consuming and prone to errors. Here, we demonstrate a full, walk-away solution for reliable and high throughput automated western blotting, combining INTEGRA's ASSIST PLUS pipetting robot and D-ONE single channel pipetting module with ProteinSimple's Simple Western Jess.

With RePlex we combined chemiluminescence and total protein detection in a single assay to prove accurate liquid handling for all reagents when setting up plates with the automated protocols. Reagents, HeLa and C2C12 lysates were prepared according to the protocol in the product insert, with lysate dilutions of 0.64, 0.32, 0.16 and 0.08 mg/ml, together with ready-to-use ERK1 primary and secondary antibodies. Each HeLa or C2C12 dilution was prepared in single tube triplicates as individual samples during plate filling.

Figure 9 shows fully automated western blot normalization of HeLa lysates (lanes 2-13), and C2C12 (lanes 14-25), in triplicate. After ERK1 detection (Figure 9a; left), the primary and secondary antibodies were removed with the RePlex reagent mix, to re-stain samples for total protein analysis (Figure 9a; right). The data sets generated were automatically analyzed, using the Simple Western software tool to visualize the uncorrected (Figure 9b; left) and corrected (Figure 9b; right) EKR1 peak target areas. All 3 replicates of each lysate showed great reproducibility while successfully normalizing EKR1 protein levels in 4 different concentrations, proving accurate liquid handling and confirming on-deck reagent/sample stability during plate set-up.

Furthermore, we performed a simple fluorescence-based detection to prove the equivalence in performance between automated and manual plate filling, by removing the luminol-peroxide mix from the VIALAB protocol for chemiluminescence detection (Chapter 4.2).

Again, reagents and HeLa lysates were prepared according to the protocol in the product insert, with sample dilutions of 0.64, 0.32, 0.16, 0.08, 0.04 and 0.00 mg/ml (0.1x sample buffer). The primary antibody was prepared by diluting 15 µl of HSP60 and 6 µl of ß-actin in 279 µl of milk-free antibody diluent. The secondary antibody was prepared by diluting 15 µl of anti-rabbit IR and 15 µl of anti-mouse NIR in 270 µl of milk-free antibody diluent.

Figure 10 a) illustrates the results of automated and manual Jess plate set-up (lane view). Wells 1-13 of rows A to D, as well as the wash buffer compartments, were filled using D-ONE and ASSIST PLUS. Wells 14-25 of rows A to D were filled manually using a single channel pipette. Both filling methods produced reliable fluorescence data when processing the first 3 dilutions of the HeLa lysates in quadruplicate (0.64 and 0.32 mg/ml) and triplicate (0.16 mg/ml), with CVs within the instrument’s specifications.

Figure 10 b) illustrates the results when performing fluorescent detection of all 6 HeLa dilutions, by preparing each dilution in 4 single tube replicates on the ASSIST PLUS deck (lane view and electropherogram showing 0.64 mg/ml replicates as representation). Again, CVs met the instrument specifications while showing high detection sensitivity for low abundance targets when automating plate filling with D-ONE and ASSIST PLUS.

Remarks

• Labware: The simple labware creation tool in the VIALAB library makes the integration of special plates easier than ever.
• VIALAB software: VIALAB programs can be easily adapted to your specific pipette, labware and protocols.
• Partial plates: Programs can be adapted at any time to a different number of samples, giving laboratories total flexibility to meet current and future demands.