23625-Mammal-c-myc-tag-co-ip-250

Pull-down is an extension of co-immunoprecipitation (Co-IP) for the capture and isolation of a target protein (i.e., the antigen) as well as its associated partners. Tag-based IP and pull-down strategies utilize a "bait" protein instead of an antibody to capture protein-interaction targets by utilizing an affinity resin to the bait protein that is biotinylated his-, GST-, c-Myc or HA-tagged.

  • More choices—different strategies to accommodate various labeled bait proteins, elution conditions, and downstream analysis needs
  • Complete—kits include all controls and lysis, binding, wash, and elution buffers
  • Fast—spin columns and collection tubes are included that shorten the protocol and minimize sample handling
  • Flexible—can scale up or down as needed using adaptable protocol
 
  Pierce Biotinylated Protein Interaction Pull-Down Kit EZ-Link Desthiobio-tinylation and Pull-Down Kit Pierce c-Myc Tag IP/Co-IP Kit Pierce HA Tag IP/Co-IP Kit Pierce GST Protein Interaction Pull-Down Kit Pierce His Protein Interaction Pull-Down Kit
Target Biotinylated proteins Biotinylated proteins c-Myc–tagged recombinant protein HA-tagged recombinant protein GST-tagged recombinant protein His-tagged recombinant protein
Base bead Streptavidin agarose resin, High-capacity streptavidin agarose resin Anti-c-Myc agarose resin Anti-HA agarose Glutathione agarose Cobalt chelate agarose resin
Binding capacity 1–3 mg/mL >10 mg/mL 102 nmol/mL >60 nmol/mL 8–10 mg/mL 10–25 mg/mL
Non-specific binding Low Low Lower Lower Lowest Lowest
Elution conditions Low pH (2.8) Buffer Mild (free biotin buffer) Low pH (2.0) buffer Non-reducing sample buffer 100 mM reduced glutathione 250 mM imidazole solution
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Effectiveness of elution options in the IP of GST-HA and GST-c-Myc from bacterial lysates.

IP results achieved with the Thermo Scientific Pierce HA IP/Co-IP Kit and c-Myc IP/Co-IP Kit using the appropriate positive control lysate provided and suggested elution options for GST-HA and GST-cMyc, respectively. The elution components are supplied with each kit. Elution #1 performed with elution buffer; Elution #2 performed with 2X nonreducing sample buffer.

a-GST-Tag-Pull-Down
b-PolyHis-Tag-Pull-Down
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 from the lysate in Lane 1 bound to an immobilized reduced glutathione support for 1 hour at 4°C. Flow-through from the lysate in Lane 1 bound to an immobilized cobalt chelate support 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 wild-type Smac (prey protein). Lysate from E. coli expressing GST-tagged BIR2 (prey protein).
6 Flow-through from the lysate in Lane 5 is allowed to interact with the immobilized bait for 1 hour at 4°C. Flow-through from the lysate in Lane 5 is allowed to interact with the 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 support with 100 mM reduced glutathione. Western blotting confirms that the minor bands observed in Lanes 9 and 11 are degradation products of GST-tagged BIR2. Elution of bait:prey complex (prepared in Lanes 1-8) from the support with 250 mM imidazole.