The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.
Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.
**This video protocol is based on an associated publication 1: High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry. Adam G. Schrum, Diana Gil, Elaine P. Dopfer, David L. Wiest, Laurence A. Turka, Wolfgang W. A. Schamel, Ed Palmer. Science’s STKE 2007 (389): pl2, June 5, 2007, [DOI: 10.1126/stke.3892007pl2]. Please click here to see this publication.
Prior to Starting, Prepare the Following Aqueous Stock Solutions:
MES Coupling Buffer: | Store at 25°C |
MES, pH 6.0 | 50 mM |
EDTA | 1 mM |
Quenching/Blocking/Storage (QBS) Buffer: | Store at 4°C |
BSA | 1% |
Sodium azide | 0.02% |
PBS, pH 7.4 | 1x |
FCM Buffer: | Store at 4°C |
Tris, pH 7.4 | 50 mM |
NaCl | 100 mM |
Sodium azide | 0.02% |
FBS | 5% |
PBS, pH 7.4 | 1x |
Prepare Immediately Prior to Use:
EDAC-MES: | Dissolve 50 mg/mL EDAC powder in MES Coupling Buffer |
Digitonin solution (2% w/v): | Dissolve digitonin powder in dH2O by heating to 95°C for 5min then cooling on ice |
Lysis Buffer: | |
Tris, pH 7.4 | 50 mM |
NaCl | 150 Mm |
Digitonin solution | 1% |
Protease Inhibitors | 1x |
Keep on ice |
1. Coupling of mAb to Beads
2. Post-nuclear Lysate Preparation and Ip
The lysis method and optimal lysis conditions will depend on the cell type and protein-protein interactions being examined. A number of protocols exist and can be modified for your application.
3. Probing of Bead-captured Protein with Fluorochrome-conjugated Antibodies
4. FCM Acquisition
The CML beads described in this protocol are 3 to 5 μm in diameter, approximately half the diameter of a quiescent mouse lymphocyte. Therefore, it may be necessary to manually increase the Forward Scatter (FSC) amp gain and the Side Scatter (SSC) voltage in order for the population of bead events to register on scale. Individual IP beads should form a single tightly clustered population. The settings and gate should be adjusted to exclude bead doublets and debris.
5. Representative Results
Figure 1. IP-FCM for TCR/CD3 multiprotein complex. T cells from the mouse strain BALB/c were lysed in 1% digitonin, and the lysate was subjected to IP-FCM using anti-CD3ε beads. The captured complexes contained significant quantities of TCR-β (purple region) and co-associated CD3-ε (green), but close to background levels (defined by the irrelevant immunoglobulin probe, pink trace) of other proteins such as Thy1.2, CD45, or H-2K(d) (brown, orange, and blue traces, respectively). See similar previously published results1-6.
Information about protein-protein interactions is highly relevant to the analysis of many cellular processes such as signal transduction, lineage maturation, cell-cycle progression, and apoptosis cascades. IP-FCM provides a quick, quantitative, and sensitive way to examine the interaction of proteins and define members of multiprotein complexes in their native conformation. Beads can be coupled and incubated with cell lysates in one day and can be probed and analyzed the next day. A 96-well plate format allows for large numbers of samples to be analyzed at one time to provide efficient data collection for statistical or screening purposes. Using fluorescent bead standards, the number of proteins captured by each bead can be estimated. Very little source material is needed for capture and detection, so limited samples and scarce analytes can still be analyzed for multiple interactions. Although IP-FCM does not require genetic engineering, epitope-tagging, denaturation, or in-vitro mixing of proteins in a non-physiological environment, it can be coupled with these and other techniques, making it a valuable and accessible tool with applicability to many biological systems.
Troubleshooting:
Many IP-FCM experiments generate useful protein interaction data the first time they are attempted. However, optimization of IP-FCM can improve antibody conjugation to beads, protein complex capture, and fluorescent probe binding.
The efficiency of IP antibody conjugation can be determined by probing coupled beads directly with an anti-immunoglobulin antibody. If this efficiency is low, increasing the concentration of antibody during the coupling reaction can allow more IP antibodies to attach to each bead. This can increase the binding capacity of the IP bead batch, resulting in enhanced capture and detection of analytes. Other primary-amine containing molecules (e.g. Tris, bovine serum albumin) should not be present during the coupling reaction, as these can compete with the mAb for bead attachment and result in low mAb coupling. If conjugation of mAb to beads proves problematic for other reasons, beads can instead be coupled to avidin/streptavidin, and biotinylated antibodies can subsequently be non-covalently bound and used for immunprecipitation.
Despite good antibody conjugation, initial detection of bead-associated fluorescence may be low. First, complex capture itself may be low. Because IP-FCM depends on the concentration of analytes, increasing the number of cells lysed per unit lysis volume can increase analyte capture and detection. Additionally, capture may be enhanced by decreasing the number of IP beads incubated with the lysate, which distributes captured complexes across fewer beads, and results in increased average fluorescence per bead when probed. Second, it is possible that access of the probe antibody to the captured complexes is sterically hindered by the immunoprecipitating antibody. In this case, the problem can be addressed by using different antibodies to capture and/or probe.
This work was supported by the Eagles Innovation Award (Fraternal Order of Eagles) and by the Mayo Foundation.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
carboxylate-modified polystyrene latex (CML) beads (5mm surfactant-free) | Reagent | Interfacial Dynamics Corporation/Molecular Probes | 2-5000 | Store at 4°C. |
Rainbow Calibration Particles | Reagent | Spherotech, Inc. | RCP-30-5A | Store at 4°C. |
2-[N-Morpholino] ethanesulfonic acid (MES) | Reagent | Sigma | M-5287 | |
1-Ethyl-3-(3-dimethylaminopropl) carbodiimide HCl (EDAC) | Reagent | Pierce Sigma |
22980 E-6383 |
Store at -20°C under desiccating conditions. |
Protease Inhibitor General Use Cocktail, containing AEBSF, Bestatin, Aprotinin, EDTA, E-64, and Leupeptin | Reagent | Sigma | P-2714 | Prepare 100x stock solution in H2O. Aliquot and store at -20°C. Handle with gloves. |
PCR plates, 96-well 0.2mL, natural, tall chimney | Reagent | Fisher | 08-408-230 | For use in place of microcentrifuge tubes during FCM staining |