Chapter Three: Pull-down Assays
Contents Page Pull-down assays overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Pull-down assay formats
GST pull-downs
Bacterial expression of both bait and prey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
Bacterial expression of bait protein. Mammalian expression of prey protein . . . . . . . . . .8
Bacterial expression of bait protein. Mammalian cell-free expression of prey protein . . .9 HaloTag-bad pull-downs
Mammalian cell expression of both bait and prey protein . . . . . . . . . . . . . . . . . . . . . . . .10
Mammalian cell-free expression of both bait and prey protein . . . . . . . . . . . . . . . . . . . .11 When to u pull-down assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Reagent requirements for GST-bad pull-downs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Reagent requirements for HaloTag pull-down assays in mammalian cells . . . . . . . . . . . . . . . . . . . . . .12 Reagent requirements for HaloTag pull-down assays using
cell-free expression systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Pull-down assays overview
Pull-down assays probe interactions between a protein of interest that is expresd as a fusion
protein (e.g., bait) and the potential interacting partners (prey).
In a pull-down assay one protein partner is expresd as a fusion protein (e.g., bait protein) in
E. coli and then immobilized using an affinity ligand specific for the fusion tag. The immobilized
bait protein can then be incubated with the prey protein. The source of the prey protein
depends on whether the experiment is designed to confirm an interaction or to identify new
rop
interactions. After a ries of wash steps the entire complex can be eluted from the affinity
support using SDS-PAGE loading buffer or by competitive analyte elution, then evaluated by
SDS-PAGE.
Successful interactions can be detected by Western blotting with specific antibodies to both the
lead on
prey and bait proteins, or measurement of radioactivity from a [35S] prey protein.
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Pull-down assay formats
GST pull-downs
Bacterial expression of both bait and prey proteins
The most commonly ud method to generate a bait protein is expression as a fusion protein contain a GST (glutathione-S-transfera) tag in E. coli. This is followed by immobilization on particles that contain
reduced glutathione which binds to the GST -tag of the fusion protein. The primary
advantage of a GST tag is that it can increa the solubility of insoluble or mi-soluble proteins expresd in E. coli.
An alternative fusion tag such as 6X
polyhistidine can also be ud to express the prey or bait protein in E. coli. Using this
format, large amounts of bait and prey protein partners can be expresd. Since both proteins are expresd in a prokaryotic
environment, interactions that require folding or modification of one or both partners may not occur.
Bacterial expression of bait protein. Mammalian expression of prey protein (Figure 3)
In this format the bait protein is expresd as a GST fusion protein in E. coli followed by immobilization on particles containing reduced glutathione.
Expression of endogenous or recombinant prey proteins in eukaryotic cells increas the probability that they will be properly modified or folded. The modifications can be critical for successful protein:protein interactions to occur.
Mammalian cell extracts containing the expresd prey protein are prepared and allowed to interact with the immobilized GST fusion bait protein.
REFERENCES
Bacterial expression of both bait and prey proteins
船期英语
1.Simader, H. et al. (2006) Nuc.Acids. Res. 34, 3968–79.
2.Phillips, P . et al. (2006)
J. Biol. Chem. 281, 4903–10.3.Reinert, L. (2006) Cancer. Res.66, 8994–9001.4.Onken, B. et al. (2006) Proc.Natl. Acad. Sci. 103, 9045–50.5.Li, H. et al. (2006) J. Biol.Chem. 281, 14748–55.
Bacterial expression of bait (GST fusions) and mammalian cell expression of prey
1.Osler, M. et al. (2005) J.Cell Sci. 118, 4667–78.
2.Ingham, R. et al. (2005) Mol.Cell. Biol. 25, 7092–106.
3.Hirst, J. et al. (2005) Mol. Biol.of the Cell 16, 2554–65.
4.Wißmüller, S. et al. (2006) Nuc.Acids Res. 34, 1735–44.
5.Chan, W. et al. (2005)
J. Biol. Chem. 25, 23741–47.
Bacterial expression of bait (polyhistidine fusions),
mammalian cell expression of prey
1.Vermeulen, M. et al. (2006) Mol.Cell. Biol. 26, 5226–36.
2.Shao, H. et al. (2006)
J. Immunol. 176, 2933–41.3.Hublitz, P . et al. (2005) Genes and Development 19, 2912–24.4.Desterro, J. et al. (2005) Mol.Biol. Cell 16, 5115–26.
Figure 3.Schematic of pull-down assay using bacterial expression of bait protein and mammalian expression of prey protein.The quence of the bait protein is cloned into a vector containing a GST tag and the appropriate elements for growth and expression li. Following expression the GST bait fusion protein is purified using glutathione particles,which bind to the GST tag.A cellular extract is prepared from mammalian cells containing the prey protein.An aliquot of the cell extract is then allowed to interact with the bound GST bait fusion protein for veral hours.After washing away non-specifically bound proteins the complex is eluted by adding reduced glutathione and analyzed by Western blotting.
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Bai t Exp r esd as GST Fusion Gel Electrophoresis Followed
by Western Blotting
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Bacterial expression of bait protein. Mammalian cell-free expression of prey protein (Figure 4)
Utilizing this format requires that the bait protein be expresd as a (GST) fusion protein in E. coli and immobilized on a support resin. Mammalian, cell-free
expression systems are ud to generate a non-fusion prey protein. Expresd prey proteins are then allowed to interact with the immobilized GST fusion bait proteins. When
using cell-free expression systems prey proteins can be expresd in 1-2 hours and ud without purification. This convenience enables the rapid characterization of veral different prey protein domains created by site-directed mutagenesis. The effects of
specific mutations on the interaction can then be evaluated.
REFERENCES (CONTINUED)
Bacterial expression of bait
protein (GST fusions) mammalian cell-free expression systems as the source of prey protein
1.Liang, S. and Lutz, C. (2006)RNA 12, 111–21.
2.Gu, X. et al. (2006) J. Bio.Chem. 281, 17882–89.
3.Cohen, R. et al. (2006)
J. Clin. Endocrinol. Metab. 91,239–47.4.Hoberg, J. et al. (2006) Mol. Cell. Biol. 26, 457–71.
arxivHaloTag pull-downs using
mammalian cell-free expression systems or mammalian cell extracts
1.Urh, M. et al. (2005) Promega Notes 92, 21–4.
2.Los, G.V . et al. (2005) Cell Notes 11, 2–6.
Figure 4.Schematic of pull-down assay using bacterial expression of bait protein and mammalian cell-free systems for the expression of prey protein.The quence of the bait protein is cloned into a vector that contains the GST tag and the appropriate elements for growth and protein expression in
E. coli. Following expression the GST bait fusion protein is immobilized using glutathione particles which bind to the GST tag.The quence of the prey protein is cloned into a vector containing the appropriate elements for
expression using a mammalian-bad cell-free expression system.An aliquot of the expresd prey protein is then allowed to interact with the bound GST bait fusion for veral hours.After washing away non-specifically bound proteins the prey is eluted and analyzed typically by Western blotting.Using an alternative procedure the prey may be expresd as [35S]-labeled protein and detected directly in the gel.
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Cell F r ee Exp r ession Bai t exp r esd as GST Fusion Gel Electrophoresis Followed
by Western Blotting
Wash and Elute Complex
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Pull-down assay formats HaloTag-bad pull-downs
Mammalian cell expression of both bait and prey proteins (Figure 5)
浣熊的英文
The properties of the HaloTag fusion protein provide some important advantages that increa the chance of successfully isolating interacting partners. First the HaloTag protein provides covalent attachment to a resin containing a HaloTag ligand (e.g., HaloLink
Resin). This covalent linkage enables
extensive washing to remove nonspecifically bound proteins, which can be problematic for most pull-down experiments.
Using this format, cells are transfected with HaloTag bait fusion protein and allowed to form complexes with endogenously
expresd prey proteins. Cells are lyd and the complexes captured by adding the HaloLink Resin. After a ries of washing steps the prey are detected by gel analysis followed by Western blotting.
Figure 5.Schematic of HaloTag pull-down assay using mammalian cells to express both the prey and
the bait proteins.The protein coding quence of the bait protein is cloned into a HaloTag vector containing the necessary elements for growth and protein expression in mammalian cells.This recombinant vector is then transfected into the appropriate mammalian cell line.The HaloTag bait fusion interacts and forms a complex with the prey protein.Whole cell extracts are then prepared.The complex is then immobilized using the HaloLink Resin which forms a covalent bond with the HaloTag fusion tag.After extensive washing of the immobilized complex to remove non-specific proteins,the prey is eluted and typically analyzed by Western blotting or mass spectrometry.
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Gel Electrophoresis Followed
by Western Blotting
Endogenous Expresd Prey Protein
Protein Interaction Complex
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Mammalian cell-free expression of both bait and prey proteins (Figure 6)
Mammalian cell-free expression systems can also be ud to express both bait and prey proteins. The u of GST or polyhistidine fusion proteins can be problematic in cell-free expression systems becau of high background. The technical issues can be resolved by expressing the bait protein as a HaloTag fusion protein.
Using this approach, the prey protein is expresd as a [35S] or fluorescent-labeled protein containing no fusion tag. The bait
protein is expresd as HaloTag fusion. The protein complex is allowed to form and then captured (using HaloLink Resin) and washed.The prey is then eluted from the complex and analyzed by gel electrophoresis.
When to u pull-down assays
Since pull-down assays can utilize prey
proteins expresd in veral different ways,such as endogenously in mammalian cells or in cell-free expression systems this technique can be ud as either a discovery or a characterization tool.
Figure 6.Schematic of pull-down assay using cell-free expression systems to express both bait and prey proteins.The protein-coding quences of the bait and prey proteins are cloned into individual vectors containing the necessary elements for expression using mammalian-bad cell-free systems.Proteins are expresd and aliquots from each reaction are mixed and the complex is allowed to form.The complex is then immobilized using the HaloLink Resin which forms a covalent bond with the HaloTag fusion tag.After extensive washing of the immobilized complex to remove non-specific proteins,the prey is eluted and typically analyzed by Western blotting.Using an alter-native procedure the prey may be expresd as [35S]-labeled protein and detected directly in the gel.
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narrative
Gel Electrophoresis Followed
by Western Blotting
Cell F r ee Exp r ession of P r ey P r o t ein
Cell F r ee Exp r ession HaloTag
Fusion Bai t P r o t ein
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Reagent requirements for GST-bad pull-downs •Glutathione particles
•2X SDS gel loading buffer •SDS polyacrylamide gel
•
Appropriate GST vector containing coding quences for bait protein •Source of prey protein (e.g., cell-free expression system, mammalian cells)•Western blotting/antibodies (if using non-labeled prey)
•[35S] methionine (if using labeled prey proteins from a cell-free expression system)
Reagent requirements for HaloTag pull-down assays in mammalian cells •Transfection reagent
•Mammalian cell line expressing prey protein
•Appropriate HaloTag vector containing coding quences for bait protein •HaloLink Resin
•Antibodies to prey and bait proteins •Western blotting reagents
Reagent requirements for HaloTag
ib课程pull-down assays using cell-free expression systems •2X SDS gel loading buffer •SDS polyacrylamide gel •Cell-free expression system
考博士的条件
•Appropriate HaloTag vector containing protein-coding quences for the bait protein
•Appropriate vector containing protein-coding quences for the prey protein •HaloLink Resin
•Western blotting (if using non-labeled prey)
•
[35S] methionine (if using labeled prey)
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