Western blot analysis of IR fluorescently labelled proteins using the G:BOX Imaging System and the Odyssey Infrared Imaging System.

doi:10.1038/protex.2011.274

Method Article

Western blot analysis of IR fluorescently labelled proteins using the G:BOX Imaging System and the Odyssey Infrared Imaging System.

https://doi.org/10.1038/protex.2011.274

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Biological techniques

Computational biology and bioinformatics

Western blot analysis

G:BOX IR6 imaging system

IR fluorescence

Odyssey Infrared imaging system

This protocol describes two imaging methods for Western blot detection of IR fluorescently labelled proteins using the CCD based G:BOX IR⁶ Imaging System and laser scanning Odyssey Infrared Imaging System. This protocol was used to detect expression of Actin and ERK proteins derived from HeLa cells. Using fluorescence-based fluorophores has several advantages over the more widely used chemiluminescent detection methods: (i) it increases sample throughput as the multiple-colour detection makes it possible to simultaneously detect two or three different proteins using different secondary antibodies (see Figure 1), (ii)multiplexing several fluorophores simultaneously can be less time consuming, (iii) near-infrared (NIR) fluorescent dyes (spectral region 650-900nm) can significantly reduce background auto-fluorescence providing enhanced detection sensitivity and quantification of proteins, (iv) many IR fluorescent dyes are stable and the membranes can be stored before scanning or imaging, (iv) some IR fluorescent dyes are competitively priced and permit the use of lower conjugate concentrations and (vi) the CCD and laser-based imaging systems uses electronic data files which allows band intensity to be varied.

• HeLa cell lysate (2.92mg/ml sample) (cultured and prepared in house)

• LDS sample buffer (4X, Invitrogen), diluted to 1X in protein sample and containing 1X Sample Reducing Agent (10X, Invitrogen)

• NuPAGE® Novex® 4-12% Bis-Tris pre-cast SDS-PAGE gel (12 well, 1.0mm) (Invitrogen)

• 1 X NuPage MOPS Running Buffer: (Invitrogen), 20X stock diluted in MilliQ-water

• Pre-stained blue protein size maker: Precision All Blue Protein Standards (Bio-Rad)

• Nitrocellulose membrane (0.45ųm, Invitrogen)

• 20X NuPAGE Transfer buffer (Invitrogen) diluted to 1X in Milli-Q water and containing 10% methanol.

• Primary antibodies

Anti-actin monoclonal (1:5000) (Abcam, UK)
Anti-ERK (1:1000) (Cell Signaling Technology)

• Secondary antibodies

Anti-mouse IgG: DyLight 680 (1:15,000) (Thermo Fisher Scientific)
Anti-rabbit IgG DyLight -800 (1:15,000) (Thermo Fisher Scientific)

• Odyssey Blocking Buffer (LI-COR)

• PBST: 0.1% Tween-20 in PBS

• XCell Sure-Lock Mini-Cell (Invitrogen)

• XCell II Blot Module (Invitrogen)

• G:BOX IR⁶ Imaging System (Syngene)

• Odyssey Infrared Imaging System (LI-COR Biosciences)

A. Western blot analysis

Dilute HeLa cell lysate (2.92mg/ml) to a concentration ranging from 29.2 -0.028µg total protein in 1X LDS sample buffer + 1X sample reducing agent and load 10µl on to a NuPAGE® Novex® 4-12% Bis-Tris pre-cast SDS-PAGE gel.
Load Precision All Blue marker (2µl) (BioRad) on to the gel. The blue protein marker bands autofluoresce in the 700nm channel and can therefore be visualised by the G:BOX IR⁶ Imaging System and the Odyssey Infrared Imaging System.
Using the XCell Sure-Lock Mini-Cell, run the gel for 30 minutes at 60V and then increase the voltage to 120V until the dye front reaches the bottom (~1 hour 30 minutes).
Prepare the nitrocellulose membrane by wetting in 1X NuPAGE transfer buffer + 10% methanol.
Prepare the transfer stack for the XCell II Blot Module according to manufacturers instructions and transfer proteins for 1 hour 15 minutes at 30V in 1X NuPAGE transfer buffer + 10% methanol.
Remove nitrocellulose membrane from the transfer stack and rinse in PBST.
Block the membrane with Odyssey Blocking Buffer for 1 hour at room temperature with gentle agitation.
Incubate the membrane overnight at 4^oC on a rocker with primary antibodies diluted in Odyssey Blocking Buffer + 0.1% Tween (mouse monoclonal anti-actin, 1:5000 and rabbit polyclonal anti-ERK, 1:1000).
Wash the membrane 4 times with PBST, 5 minutes each wash.
Incubate the membrane with secondary antibodies diluted in Odyssey Blocking Buffer + 0.1% Tween for 1 hour at room temperature in the dark with gentle agitation.
Wash the membrane 3 times with PBST, 5 minutes each wash.
Finally, rinse the membrane with PBS alone before scanning or using the imaging system.

Visualisation

G:BOX IR⁶ system settings for DyLight 680 are an Epi Red lighting and a Syngene 705M filter and for DyLight 800 are Epi LED IR740 lighting and a Syngene LY800 filter, 4x4 binning factor for both dyes.
Odyssey system settings are preset: membrane, resolution: 169 mm, quality: medium, focus offset: 0.0, intensity 4.5 for DyLight 680 and intensity 5 for DyLight 800.
DyLight 680 is detected as red bands and DyLight 800 is detected as green bands 800nm window. The G:BOX IR⁶ and Odyssey imaging system can successfully multiplex between the DyLight 680 and DyLight 800 using the stated imaging conditions. The results from both systems show comparable sensitivity (Figure 1).

Determining linear range and limit of detection (LOD)

To determine the linear range and LOD of each of the DyLight dyes, plot the amount of actin and ERK proteins loaded onto the gel against the Integrated Intensity (K counts mm²) for the Odyssey system using Odyssey software (Figure 2a and 3a), and against raw volume (Signal Intensity) for G:BOX IR⁶ using the GeneTools software (Figure 2b and Figure 3b).
Using the Odyssey and G:BOX IR⁶ systems the DyLight 680 was linear over the following range (7.3-0.22µg) series dilution used in this experiment (Figure 2a and 2b).
The DyLight 800 was linear with Odyssey and G:BOX IR⁶ systems over the dilution series used in this experiment. To compare the linearity of the data and to determine LOD ERK p44 (DyLight 800) was used.
The Odyssey system produces a linear range for ERK p44 with R² value of 0.98 and has a LOD of 0.11µg (Figure 3).The G:BOX IR⁶ system produces a linear range for ERK p44 with R² value of 0.98. The LOD with the G:BOX IR⁶ system is 0.11µg (Figure 3).

About 17.5 hours

Sample preparation; 1 hour, SDS-PAGE; 1.5 hours, Transfer; 1 hour 15 minutes, Western blotting; 14 hours.

Scanning and analysis using the Odyssey scanner 3 minutes 42 seconds using the G:BOX IR⁶ 2 minutes 20 seconds.

High background:

a) Too much primary and/or secondary antibodies used. If necessary, perform a titration experiment with the primary antibody to determine the optimal dilution.

Proteins will be detected as green or red bands on the membrane.

Pre-stained blue size marker: red, DyLight 800: green, DyLight 680: red.

Mathews ST, et al. Imaging Systems for Westerns: chemiluminescence vs Infrared detection. Methods Mol Biol. 536,499-513 (2011).

The authors declare no competing financial interests.

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Western blot analysis of IR fluorescently labelled proteins using the G:BOX Imaging System and the Odyssey Infrared Imaging System.

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Western blot analysis of IR fluorescently labelled proteins using the G:BOX Imaging System and the Odyssey Infrared Imaging System.

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Abstract

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Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Additional Declarations

Associated Publications

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