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Overview of Pierce qIP Protein Interaction Kits

Learn about quantitative IP assays based on luciferase bioluminescence.

Pierce qIP Protein Interaction KitsThermo Scientific Pierce qIP Protein Interaction Kits use anti- HA or anti-Myc beads (agarose or magnetic) and a sensitive luciferase assay system to co-immunoprecipitate (co-IP) and quantify interactions between epitope-tagged and Tluc-tagged protein pairs expressed in mammalian cells. The quantitative immunoprecipitation (qIP) system depends on TurboLuc luciferase enzyme (Tluc) to accurately and precisely reflect the abundance of a specific co-IP product without time-consuming gel electrophoresis, Western blot and band densitometry steps.

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Overview of Protein-Protein Interaction Analysis

 

 

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Pierce qIP Protein Interaction Kits

The Pierce Quantitative Immunoprecipitation (qIP) Assay System

Sensitive Luciferase Assay

Pierce qIP Assays depend on a sensitive luciferase assay system for quantitation. Pierce TurboLuc™ (Tluc) is a novel intracellular luciferase derived from the marine copedod Metridia luciferase family. The Tluc gene was developed using directed mutagenesis for optimal performance and codon optimized for mammalian cell expression. Tluc is an ATP-independent enzyme that utilizes the substrate coelenterazine to produce intense bioluminescence light. Tluc is smaller (18 kDa) and much brighter than other commonly used luciferases, including firefly and Renilla, making it the ideal luciferase for bioluminescent assays. The intense bioluminescence greatly enhances sensitivity of Tluc luciferase-based assays, enabling detection of very minute amounts of luciferase (signal) in qIP assays. In addition, the Tluc luciferase gene is codon optimized for mammalian cell expression.

Tag-based Protein Expression and Co-IP

To test the interaction of two proteins of interest (X and Y) in this assay system, genes for X and Y must be cloned into two separate expression vectors:

  • Gene X into an epitope-tagged vector (HA-tag or Myc-tag)
  • Gene Y into Tluc-tagged vector

Pierce qIP Kits include multiple cloning site (MCS) expression vectors for this purpose (C-terminal epitope-tag vector and N-terminal Tluc vector). Other qIP expression vectors are also available.

Upon co-transfection into mammalian cells, the vectors express the X and Y proteins, which then interact based on treatment conditions. Next, the cells are lysed, and the target protein-complex is immunoprecipitated using anti-epitope-tag beads. Finally, the co-IP product (protein Y) is quantified by measuring Tluc luciferase activity.
In addition to supplying epitope and Tluc cloning vectors, kits also include prepared positive and negative control vectors that enable normalization of results.

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Buffers and Tluc Assay Reagents for Pierce qIP Assays

Expression Vectors for Pierce qIP Assays

Pierce qIP Assay Concept
Schematic of qIP assay mechanism and resulting data. This example uses HA as the epitope tag (vs. Myc) and anti-HA magnetic beads (vs. agarose resin). In the test condition (left), Tluc-tagged protein Y co-immunoprecipitates via its interaction with HA-tagged protein X. The resulting luminescence signal is directly proportional to the amount of protein Y bound to protein X (i.e., the abundance of the X-Y protein interaction). In the negative control (right), Tluc-tagged protein Y does not interact with HA-tagged RFP (red fluorescent protein) and is not pulled down, resulting in a measure of the background luminescence signal (noise). The protein interaction is represented by the normalized signal-to-noise ratio.

 


The Pierce qIP Assay Procedure

The default procedure is designed for use with mammalian cells grown in 6-well tissue culture plates. Transfection reagents, media and other components for cell culture are not included in the kits, nor are trypsin for cell release and protease inhibitor for cell lysis. However, optimized qIP reagents are included in the kits to prepare lysis and wash buffers. Kits and standalone components are available to perform the procedure for HA- or Myc-tagged IP using either agarose resin or magnetic beads. One salient feature of the assay system is that there is no elution step; washed resin slurry or bead suspension (still bound to the target protein complex) is transferred directly to a white or black microplate for measurement in a luminometer.

Schematic of the qIP assay procedure. In two separate wells of a 6-well tissue culture plate, pairs of vectors encoding the epitope-tagged and Tluc-tagged proteins for the test (X|Y) and negative control (RFP|Y) conditions are co-transfected into mammalian cells. After 24 hours, cells are collected, lysed and centrifuged to generate clear total lysate. The total lysate is incubated with anti-epitope affinity beads in for approximately 3 hours. The beads are washed, resuspended in wash buffer, and an aliquot of the suspension is transferred to a 96-well plate (triplicates per sample). Luciferase substrate solution is added to each well and the plates are incubated for 10 minutes. Relative light units (RLU) are measured with a luminometer.

 

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Agarose Kit for HA Tag

Agarose Kit for Myc Tag

Magnetic Kit for HA Tag

Magnetic Kit for Myc Tag

Highlights of the Pierce qIP System

Highlights:

  • Quantitative – integrated luciferase assay allows direct measurement of co-IP products, and control system ensures accuracy and normalization
  • Sensitive – bright bioluminescence signal produced by TurboLuc luciferase with coelenterazine substrate allows detection of even weak interactions
  • Time-saving – because final read-out is relative luminescence units (RLU) on a luminometer, elution steps and post-IP Western blotting are not required
  • Simple and fast – pull-down (co-IP) incubation and three quick washing steps done in spin columns (agarose resin kits) or microcentrifuge tubes (magnetic bead kits)
  • Robust – vectors, kit and entire qIP method are compatible with many different mammalian cell lines including 293T, HEK293, NIH3T3, CHO-K1
  • Versatile – the assay platform is highly customizable:
    • Additional cloning vectors are available for N- and C-terminal fusion;
    • HA- and c-Myc tag kits available in both agarose and magnetic bead formats;
    • Assay reagents and buffers are available separately to facilitate experiments at various scales once initial cloning is completed
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Pierce qIP Protein Interaction Kits

Browse all Pierce qIP Protein Interaction Kits


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