Pierce GST and His Tag Protein Interaction Pull-Down Kits
Purify proteins that interact with GST- or His-tagged fusion proteins.
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.
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.
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:
Chai, J., Wu, J.-W., Kyin, S., Wang, X. and Shi, Y. (2000). Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature406, 855-862.
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. Cell64, 521-532. (Discusses GST-tag pull-down assay)
Soutoglou, E., Katrakili, N. and Talianidis, I. (2000). Acetylation regulates transcription factor activity at multiple levels. Mol. Cell5, 745-751. (Discusses His-tag pull-down assay)
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.
Mori, Y. et al. (2003). J. Virol.77(8), 4992-4999.
Mori, Y. et al. (2004). J. Virol.78(9), 4609-4616.
