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Pierce Agarose qIP Protein Interaction Kit, Tluc and HA Tags


An HA-tag co-IP system with integrated luciferase assay quantitation.

Pierce Agarose qIP Protein Interaction Kit, Tluc and HA Tags

This Thermo Scientific Pierce qIP Protein Interaction Kit uses anti-HA agarose beads and a sensitive luciferase assay system to pull down (co-IP) and measure interactions between HA-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. To test the interaction of two proteins X and Y, the respective genes are cloned into two separate expression vectors supplied in the kit (gene X into HA-tagged vector, and gene Y into Tluc-tagged vector). 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-HA agarose beads. Finally, the co-IP product (protein Y) is quantified by measuring Tluc luciferase activity. In addition to supplying the HA and Tluc cloning vectors, the kit also includes prepared positive and negative control vectors that enable normalization of results.

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 washing steps carried out in supplied microcentrifuge spin columns (7 second per each washing step)
  • 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

Includes:

  • Kit contains Tluc-fusion and HA-tag pCMV multiple cloning site vectors, positive and negative control plasmids, anti-HA agarose resin, qIP assay buffers, Tluc assay reagents, spin columns and collection tubes.

Requires:

  • In vitro transfection reagent (e.g., Part No. R0533)
  • Protease inhibitors (e.g., Part No. 78437)
  • 6-well tissue culture plate
  • 96-well microplate (black or white) compatible with luminometer
  • Luminometer

Applications:

  • Measure protein-protein interaction before and after treatment of cells with growth factors (e.g., PDGF, EGF, TNFα, TGFβ)
  • Measure epigenetic modification dependent protein-protein interaction (acetylation or methylation of proteins, such as histones)

Product Details:

Pierce qIP assay mechanism for quantitative immunoprecipitation
Schematic of qIP assay mechanism and resulting data. 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 essentially zero background luminescence signal (noise). The protein interaction is represented by the normalized signal-to-noise ratio.

 

Expression vectors for qIP experiments. The kit includes two multiple cloning site (MCS) vectors for preparation of plasmids to express test proteins X and Y. Prepared BAD and Bcl-xL plasmids are included for use as a positive control condition. More importantly, prepared HA-tagged red fluorescent protein (RFP) plasmid is included to produce a negative control condition, which is used in every experiment to normalize signal-to-noise.
Cloning
Vector
Prepared
Plasmid
Expressed
Protein
Experiment
Condition
pCMV MCS C-HA pCMV X C-HA HA.X Test Interaction:
X|Y
pCMV MCS N-Tluc pCMV Y N-Tluc Y.Tluc
-- pCMV BAD C-HA HA.BAD

Positive Control:
BAD|Bcl-xL

-- pCMV Bcl-xL N-Tluc Bcl-xL.Tluc
-- pCMV RFP C-HA HA.RFP

Negative Control:
RFP|Y
RFP|Bcl-xL

 

Pierce qIP Assay Kit protocol for quantitative immunoprecipitation
Schematic of the qIP assay procedure. In two separate wells of a 6-well tissue culture plate, pairs of vectors encoding the HA-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 Pierce Anti-HA Agarose in a spin column with rotation for approximately 3 hours. The Pierce Anti-HA Agarose Resin is washed and resuspended in wash buffer. The resuspended resin slurry 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.

 

More Product Data

General References:

Related Resources:

Overview of Pierce qIP Protein Interaction Kits
Tech Tip #77: Cloning guide for Pierce qIP Protein Interaction Kits

Related Products:

Pierce qIP Protein Interaction Kits and Reagents
Standard HA Tag and Myc Tag IP/Co-IP Kits
Luciferase Assay Kits and Reagents


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
82032 Agarose qIP Protein Interaction Kit, Tluc and HA Tags
Formulation: Multi-component kit
Sufficient For: 25 qIP reactions involving HA-tagged protein

Kit Contents:
pCMV MCS C-HA Vector, 10µg
pCMV MCS N-Tluc Vector, 10µg
pCMV RFP C-HA Vector, 10µg
pCMV BAD C-HA Vector, 10µg
pCMV Bcl-xL N-Tluc Vector, 10µg
Pierce Anti-HA Agarose, 2 x 0.5mL slurry
Pierce qIP Protein Interaction Assay Buffer L (1X), 35mL
Pierce qIP Protein Interaction Assay Buffer D (10X), 12.5mL
Spin Columns – Screw Cap, 27 columns
Collection Tubes, 2mL, 27 tubes
Pierce qIP Protein Interaction Tluc Assay Buffer, 10mL
Coelenterazine Substrate (100X), 2 x 50µL
25-rxn kit Product Instructions for product #82032 Agarose qIP Protein Interaction Kit, Tluc and HA Tags MSDS for product #82032 Agarose qIP Protein Interaction Kit, Tluc and HA Tags $505.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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