Update profile
    Change password
    Sign out

Pierce GST and His Tag Protein Interaction Pull-Down Kits


Purify proteins that interact with GST- or His-tagged fusion proteins.

Pierce GST Tag and His Tag Protein Interaction Pull-Down Kits

Pierce GST and His Tag Protein Interaction Pull-Down Kits contain the necessary components to capture and purify proteins that interact with GST- or His-tagged fusion proteins, respectively.

You provide the tagged fusion protein as the "bait" and the cells expressing the putative protein interaction target ("prey"), and the Pull-Down Kits provide everything else: cell lysis buffer, microcentrifuge spin columns, tag-specific affinity resin (agarose beads) and optimized buffers and protocol. The Pull-Down Kits are designed to teach the method to first-time users and to increase ease-of-use, convenience and reproducibility for experienced researchers. The procedure is conceptually simple.

Highlights:

  • GST pull-down (Product No. 21516) – purifies protein interactors of any GST-tagged fusion protein
  • 6xHis pull-down (Product No. 21277) – purifies protein interactors of any His-tagged fusion protein
  • Complete kits – provide all components and detailed protocol for purifying protein:protein interactions
  • No special equipment needed – use common laboratory equipment and reagents (e.g., microcentrifuge)
  • Convenient – microcentrifuge spin columns facilitate simple and efficient manipulation of agarose beads, including simple processing of multiple samples
  • Flexible – instructions include protocols for bait and prey proteins from different sources

Product Details:

Procedure summary for Pierce GST-tag Protein Interaction Pull-Down Kit and His-tag Protein Interaction Pull-Down Kit
Procedure summary for GST Tag and His Tag Pull-Down Kits. "Pull-down" is a small-scale affinity purification technique similar to immunoprecipitation (IP), except that the antibody function of is replaced by some other affinity system. In this case, the affinity system is either a GST-tagged protein that can be captured by glutathione agarose beads or a His-tagged protein that can be captured by metal chelate (cobalt) agarose beads. The fusion-tagged protein acts as the "bait" to capture a putative binding partner (i.e., the "prey"). In a typical pull-down assay, the immobilized bait protein is incubated with a cell lysate. After the prescribed washing steps, the 'interactors" are selectively eluted for analysis in-gel or by Western blot.

A. B.
GST-tag Protein Interaction Pull-Down Kit results His-tag Protein Interaction Pull-Down Kit results
SDS-PAGE analysis of GST- and His-tagged protein interaction pull-down experiments performed with the Pierce Pull-Down Kits. Gels were stained with Pierce Silver Stain (Product No. 24612). Lanes represent various control, flow-through, wash and elution fractions, as detailed in Table 1 below.

Detailed description of lanes in the figure above.
Lane A. GST-Tag Pull-Down B. PolyHis-Tag Pull-Down
1 Lysate from E. coli expressing GST-tagged BIR2 (bait protein). Lysate from E. coli expressing 9xHis-tagged wild-type Smac (bait protein).
2 Flow-through lysate after binding to glutathione agarose for 1 hour at 4° C. Flow-through lysate after binding to cobalt agarose for 1 hour at 4° C.
3 Wash #1 of the support. Wash #1 of the support.
4 Wash #2 of the support. (Washes 3-5 not shown.) Wash #2 of the support. (Washes 3-5 not shown.)
5 Lysate from E. coli expressing 9xHis-tagged Smac (prey protein). Lysate from E. coli expressing GST-tagged BIR2 (prey protein).
6 Flow-through prey lysate after incubation with immobilized bait for 1 hour at 4° C. Flow-through prey lysate after incubation with immobilized bait for 1 hour at 4° C.
7 Wash #1 of the support. Wash #1 of the support.
8 Wash #2 of the support. (Washes 3-5 not shown.) Wash #2 of the support. (Washes 3-5 not shown.)
9 Bait control. Bait treated as described in Lanes 1-8 and subsequently eluted. No prey added – just binding buffer. Bait control. Bait treated as described in Lanes 1-8 and subsequently eluted. No prey added – just binding buffer.
10 Prey control. Prey treated as described in Lanes 1-8 and subsequently eluted. No bait added – just binding buffer. Prey control. Prey treated as described in Lanes 1-8 and subsequently eluted. No bait added – just binding buffer.
11 Elution of bait:prey complex (prepared in Lanes 1-8) from the agarose beads with 100mM reduced glutathione. Elution of bait:prey complex (prepared in Lanes 1-8) from the agarose beads with 250mM Imidazole.

Applications:

  • Discover a new protein:protein interaction from a cell lysate
  • Confirm a putative interaction from a cell lysate or with a previously purified protein
  • Extract protein:protein interaction information from in vitro transcription/translation lysates

GST Tag Protein Interaction Pull-Down Kit Highlights:

  • Binds approx. 8mg of GST-tagged fusion protein per ml of resin
  • Gentle elution conditions do not denature interacting proteins
  • Protocols included for use with bait and prey proteins from many different sources

His Tag Protein Interaction Pull-Down Kit Highlights:

  • Binds 10 to 25mg of histidine-tagged fusion protein per ml of resin
  • Cobalt chelate resin is more specific for histidine-tagged fusion proteins than nickel resins, resulting in less nonspecific binding
  • Protocols included for use with bait and prey proteins expressed from a variety of sample types

References:

  1. Chai, J., Wu, J.-W., Kyin, S., Wang, X. and Shi, Y. (2000). Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 406, 855-862.
  2. Kaelin, W.G., Jr., Pallas, D.D., DeCaprio, J.A., Kaye, F.J. and Livingston, D.M. (1991). Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 64, 521-532. (Discusses GST-tag pull-down assay)
  3. Soutoglou, E., Katrakili, N. and Talianidis, I. (2000). Acetylation regulates transcription factor activity at multiple levels. Mol. Cell 5, 745-751. (Discusses His-tag pull-down assay)
  4. Sambrook, J. and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual 3rd Edition. Chapter 18: Protein Interaction Technologies, Protocol #3: Detection of Protein-Protein Interactions using the GST Fusion Protein Pull-Down Technique. Cold Spring Harbor Laboratory Press: Plainview, NY.
  5. Mori, Y. et al. (2003). J. Virol. 77(8), 4992-4999.
  6. Mori, Y. et al. (2004). J. Virol. 78(9), 4609-4616.

Related Resources:

Protein Interaction Pull-Down Kits FAQ

Related Products:

Co-Immunoprecipitation Kit
Biotinylated Protein Interaction Pull-Down Kit
Pierce Spin Cups and Columns
Cobalt Agarose Resin
Glutathione Agarose Resin


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
21516 Pierce GST Protein Interaction Pull-Down Kit
Formulation: Multi-component kit
Sufficient For: At least 25 pull-down reactions, each using 25µL of resin

Kit Contents:
Glutathione Agarose, 750µL
Pull-Down Lysis Buffer, 250mL
Glutathione, 1g
TBS Pack (makes 500mL), 1 pack
Pierce Spin Columns and Accessories, 27 columns
Microcentrifuge Collection Tubes, 100 tubes
25-rxn kit Product Instructions for product #21516 Pierce GST Protein Interaction Pull-Down Kit MSDS for product #21516 Pierce GST Protein Interaction Pull-Down Kit Certificate of Analysis for product #21516 Pierce GST Protein Interaction Pull-Down Kit $350.00

Add to cart
21277 Pierce His Protein Interaction Pull-Down Kit
Formulation: Multi-component kit
Sufficient For: At least 25 pull-down reactions, each using 25µL of resin

Kit Contents:
Cobalt Chelate Agarose Resin, 750µL
Pull-Down Lysis Buffer, 250mL
Imidazole Solution (4M), 5mL
TBS Pack (makes 500mL), 1 pack
Pierce Spin Columns and Accessories, 27 columns
Microcentrifuge Collection Tubes, 100 tubes
25-rxn kit Product Instructions for product #21277 Pierce His Protein Interaction Pull-Down Kit MSDS for product #21277 Pierce His Protein Interaction Pull-Down Kit Certificate of Analysis for product #21277 Pierce His Protein Interaction Pull-Down Kit $346.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

Share This Page
 
 

Follow Us
  Email Sign-up  Email Announcements

 



Antibodies  |   Molecular Biology   |   Cell Biology   |  Thermo Scientific  |   * Trademarks  |   Privacy Statement
© 2014 Thermo Fisher Scientific Inc.   |   3747 N Meridian Rd, Rockford, IL USA 61101   |   1-800-874-3723  or  815-968-0747