Protein Analysis, Modification and Interaction
Protein interaction - Pull-down assays
PolyHis protein interaction pull-down kit, Thermo Scientific Pierce
PN
Identifies protein interaction complexes involving His-tagged proteins.
Experiment validates kit specificity and function. Lysate samples,
washes, controls and final eluted products were analysed by SDS-PAGE and
then silver-stained (PN24612). Upper and lower bands in lane 11 are the
purified prey protein and bait protein, respectively. See accompanying table
for details.
•
Binds 10mg to 25mg of His-tagged fusion protein per mL of resin
•
Cobalt chelate resin is more specific for histidine tags than nickel chelate resins, resulting in less non-specific
binding
•
Instructions include protocols for bait and prey proteins from different sources
The Thermo Scientific Pierce PolyHis protein interaction pull-down kit contains the necessary components to capture
and purify proteins that interact with polyhistidine-tagged fusion proteins.
You provide the tagged fusion protein (“bait”) and cells expressing the putative protein interaction target (“prey”), and
the pull-down kit provides everything else: cell lysis buffer, microcentrifuge spin columns, metal-chelate affinity resin
(agarose beads), optimised binding and elution buffers, and a detailed protocol.
Details of kit validation experiment (see image)
Lane
Description of sample
1
Lysate from E. coli expressing 9xHis-tagged Smac (bait protein)
2 to 4
Flow-through after bait binding (2) and subsequent washes (3 to 4)
5
Lysate from E. coli expressing BIR2 (prey protein)
6 to 8
Flow-through after prey binding (6) and subsequent washes (7 to 8).
9
Control (no prey added). Bait treated as in Lanes 1 to 8 and then eluted.
Bands is the His-tagged bait protein
10
Control. (no bait added). Prey treated as in Lanes 1 to 8 and then eluted
11
Elution of bait:prey complex. Top band is purified prey protein
Acknowledgment
We gratefully acknowledge Dr. Yigong Shi of Princeton University for supplying the GST-BIR2- and 9xHis Smac/DIABlOexpressing clones. Dr. Shi’s laboratory is engaged in research aimed at understanding the structural and molecular
mechanisms involved in tumorigenesis and apoptosis
Step 1. Immobilise the fusion-tagged
“bait” from the lysate. Wash.
Agarose
Bead
Step 2. Bind “prey” protein to
immobilized “bait” protein. Wash.
Step 3. Elute protein:protein interaction complex.
Prey
Bait Protein Protein
Bait Protein
Affinity Ligand
(Co2+ Chelate)
Bait Protein
Prey
Protein
Lysate containing
His-tagged bait protein
Lysate containing
prey protein
Imidazole
Prey
Bait Protein Protein
Bait Protein
Prey
Bait Protein Protein
Displaced interacting complex
= 6xHis Fusion Tag
Protocol summary for Thermo Scientific PolyHis protein interaction
pull-down kit.
1078
Catalogue No
Description
PN21277
PolyHis protein interaction pull-down kit
Sufficient materials for performing 25 pull-down assays using a polyhistidine-tagged
protein as the bait. Includes:
Immobilised cobalt chelate resin, 750µL
Lysis buffer, 250mL
4M Imidazole stock solution, 5mL
Tris buffered saline, 1 pack (500mL)
Spin columns, x 27
Microcentrifuge tubes, 100 x 2mL
Price
224.00